Methods of treating immune disorders using pd-1 binding proteins

ABSTRACT

Provided herein are methods for managing, treating, or preventing immune disorders, such as autoimmune diseases, using proteins that specifically bind to Programmed Death-1 (PD-1) and modulate the expression and/or activity of PD-1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/396,736, filed Sep. 19, 2016, the disclosure of which is incorporated by reference herein in its entirety.

1. FIELD

Provided herein are methods for managing, treating, or preventing immune and inflammatory disorders, such as autoimmune diseases, using proteins that specifically bind to Programmed Death-1 (PD-1) and modulate the expression and/or activity of PD-1.

2. SUMMARY

The present disclosure provides methods of managing, preventing, or treating an immune disorder in a subject comprising administering to a subject a therapeutically effective amount of proteins that bind to PD-1 (e.g., human PD-1, SEQ ID NO:43), including binding proteins such as antibodies that bind to PD-1. Such binding proteins, including antibodies, can bind to a PD-1 polypeptide, a PD-1 fragment, and/or a PD-1 epitope. Such binding proteins, including antibodies, can be agonists (e.g., induce PD-1 ligand-like signaling). In some embodiments, the binding proteins do not compete with PD-1 ligand (e.g., PD-L1 and PD-L2) for the interaction with PD-1 (e.g., a non-blocking antibody).

The present disclosure also provides, in certain embodiments, methods of managing, preventing, or treating an immune disorder in a subject comprising administering to a subject a therapeutically effective amount of binding proteins, including antibodies or fragments thereof, that (i) bind to human PD-1, (ii) induce PD-1 ligand-like signaling, and (iii) do not compete with PD-L1 and/or PD-L2 for the interaction with PD-1.

In some embodiments, a binding protein (e.g., an anti-PD-1 antibody) for use in the methods provided herein comprises six complementarity determining regions (CDRs) or fewer than six CDRs. In other embodiments, a binding protein (e.g., an anti-PD-1 antibody) comprises one, two, three, four, five, or six CDRs selected from heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, light chain variable region (VL) CDR1, VL CDR2, and/or VL CDR3. In certain embodiments, a binding protein (e.g., an anti-PD-1 antibody) comprises one, two, three, four, five, or six CDRs selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a monoclonal antibody designated as PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 as described herein, or a humanized variant thereof. In some embodiments, a binding protein (e.g., an anti-PD-1 antibody) further comprises a scaffold region or framework region (FR), including a VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3, and/or VL FR4 of a human immunoglobulin amino acid sequence or a variant thereof.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof binds to an epitope of human PD-1 recognized by an antibody comprising a light chain variable region having an amino acid sequence of SEQ ID NO:8 and a heavy chain variable region having an amino acid sequence of SEQ ID NO:13.

In other embodiments of the methods, the antibody or antibody fragment thereof competes for the binding to human PD-1 with an antibody comprising a light chain variable region having an amino acid sequence of SEQ ID NO:8 and a heavy chain variable region having an amino acid sequence of SEQ ID NO:13.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a VL comprising VL CDR1, VL CDR2, and VL CDR3 of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 as set forth in Table 1.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a VH comprising VH CDR1, VH CDR2, and VH CDR3 of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 as set forth in Table 2.

In still other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises:

-   (a) a VL comprising VL FR1, VL FR2, VL FR3, and VL FR4 of any one of     antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6     as set forth in Table 3; and -   (b) a VH comprising VH FR1, VH FR2, VH FR3, and VH FR4 of any one of     antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6     as set forth in Table 4.

In certain embodiments of the methods, the VL CDR1, VL CDR2, and VL CDR3 of the antibody or antigen-binding fragment thereof comprise amino acid sequences of SEQ ID NOS:1, 2, and 3, respectively, and the VH CDR1, VH CDR2, and VH CDR3 of the antibody or antigen-binding fragment thereof comprise amino acid sequences of SEQ ID NOS:4, 5, and 6, respectively.

In yet another embodiment of the methods, the VL CDR1, VL CDR2, and VL CDR3 of the antibody or antigen-binding fragment thereof comprise amino acid sequences of SEQ ID NOS:7, 2, and 3, respectively, and the VH CDR1, VH CDR2, and VH CDR3 of the antibody or antigen-binding fragment thereof comprise amino acid sequences of SEQ ID NOS:4, 5, and 6, respectively.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO:8. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO:9. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO:10. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO:12. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO:13. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:8; and (b) a VH comprising an amino acid sequence of SEQ ID NO: 11.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:9; and (b) a VH comprising an amino acid sequence of SEQ ID NO: 11.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:10; and (b) a VH comprising an amino acid sequence of SEQ ID NO: 11.

In one embodiment of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:8; and (b) a VH comprising an amino acid sequence of SEQ ID NO:12.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:9; and (b) a VH comprising an amino acid sequence of SEQ ID NO:12.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:10; and (b) a VH comprising an amino acid sequence of SEQ ID NO:12.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:8; and (b) a VH comprising an amino acid sequence of SEQ ID NO:13.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:9; and (b) a VH comprising an amino acid sequence of SEQ ID NO:13.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:10; and (b) a VH comprising an amino acid sequence of SEQ ID NO:13.

In some embodiments, the amino acid sequence of the VL comprises one or more conservative modifications thereof. In some embodiments, the amino acid sequence of the VH comprises one or more conservative modifications thereof. In some embodiments, the amino acid sequence of the VL and the VH comprises one or more conservative modifications thereof.

In some embodiments of the methods, the antibody comprises a human IgG1 Fc region. In other embodiments, the antibody comprises a variant human IgG1 Fc region.

In one embodiment of the methods, the antibody comprises a human IgG1-K322A Fc region.

In some embodiments of the methods, the antibody comprises a human IgG4 Fc region. In other embodiments, the antibody comprises a variant human IgG4 Fc region.

In another embodiment of the methods, the antibody comprises a human IgG4P Fc region.

In still another embodiment of the methods, the antibody comprises a human IgG4PE Fc region.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof further comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof further comprises a heavy chain Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:36-40.

In yet another embodiment of the methods, the antibody or antigen-binding fragment thereof further comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41; and a heavy chain Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:36-40.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO:31.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:32.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:32.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:33.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:33.

In one embodiment of the methods, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:34.

In yet another embodiment of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:34.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:35.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:35.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one of residues 100-109 within an amino acid sequence of SEQ ID NO:42.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one of residues 100-105 within an amino acid sequence of SEQ ID NO:42.

In particular embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one residue selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to two or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to three or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to four or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In one embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to five or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to six or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In yet another embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to seven or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In still another embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to eight or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to nine or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to all ten residues from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In one embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to N33 within an amino acid sequence of SEQ ID NO:42.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to T51 within an amino acid sequence of SEQ ID NO:42.

In a particular embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to S57 within an amino acid sequence of SEQ ID NO:42.

In one specific embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to L100 within an amino acid sequence of SEQ ID NO:42.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to N102 within an amino acid sequence of SEQ ID NO:42.

In other embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to G103 within an amino acid sequence of SEQ ID NO:42.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to R104 within an amino acid sequence of SEQ ID NO:42.

In yet another embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to G103 and R104 within an amino acid sequence of SEQ ID NO:42.

In still another embodiment of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to D105 within an amino acid sequence of SEQ ID NO:42.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to H107 within an amino acid sequence of SEQ ID NO:42.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to S109 within an amino acid sequence of SEQ ID NO:42.

In one embodiment of the methods, the epitope of human PD-1 is distinct from the PD-L1 binding site. In another embodiment, the epitope of human PD-1 is distinct from the PD-L2 binding site. In a specific embodiment, the epitope of human PD-1 is distinct from both the PD-L1 binding site and the PD-L2 binding site.

In an embodiment of the methods, the antibody or antigen-binding fragment thereof specifically binds to human PD-1 and/or monkey PD-1 (for example, cynomolgus monkey), but not rodent PD-1.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof has attenuated antibody dependent cellular cytotoxicity (ADCC) activity. In other embodiments, the antibody or antigen-binding fragment thereof has attenuated complement dependent cytotoxicity (CDC) activity. In some embodiments, the antibody or antigen-binding fragment thereof has attenuated ADCC and/or attenuated CDC activity.

In one aspect, provided herein is a method of managing, preventing, or treating an immune disorder in a subject, comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to an epitope of human PD-1, wherein the antibody or antigen-binding fragment thereof: (a) attenuates T cell activity; and/or (b) downregulates PD-1 expression on the surface of T cells.

In one embodiment of the methods, the antibody attenuates T cell activity. In another embodiment, the antibody downregulates PD-1 expression on the surface of T cells.

In certain embodiments, the attenuation of T cell activity is measured by a T cell effector function.

In some embodiments of the methods, the attenuation of T cell activity is measured by inhibition of cytokine production.

In certain embodiments of the methods, the cytokine that is inhibited by the antibody or antigen-binding fragment thereof comprises IL-2, IL-17, and/or IFN-γ. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, 1-22, IL-23, GM-CSF, IFN-γ, and TNF-α. In certain embodiments, the cytokine is IL-1. In some embodiments, the cytokine is IL-2. In other embodiments, the cytokine is IL-6. In another embodiment, the cytokine is IL-12. In some other embodiments, the cytokine is IL-17. In yet other embodiments, the cytokine is IL-22. In still other embodiments, the cytokine is IL-23. In some embodiments, the cytokine is GM-CSF. In other embodiments, the cytokine is IFN-γ. In yet other embodiments, the cytokine is TNF-α. In certain embodiments, the cytokine is IL-2 and IL-17. In some embodiments, the cytokine is IL-2 and IFN-γ. In yet other embodiments, the cytokine is IL-17 and IFN-γ. In still other embodiments, the cytokine is IL-2, IL-17, and IFN-γ. Other combinations of two, three or more of the above-mentioned cytokines are also contemplated.

In some embodiments of the methods, the attenuation of T cell activity is measured by inhibition of T cell proliferation.

In certain embodiments of the methods, the attenuation of T cell activity is measured by upregulation of expression of an inhibitory receptor on the surface of T cells, wherein the inhibitory receptor is not PD-1. In some embodiments, the inhibitory receptor is selected from the group consisting of LAG-3, CTLA-4, and TIM-3. In one specific embodiment, the inhibitory receptor is LAG-3. In another specific embodiment, the inhibitory receptor is CTLA-4. In yet another specific embodiment, the inhibitory receptor is TIM-3.

In some embodiments of the methods, the attenuation of T cell activity is measured by inhibition of T cell recall response.

In certain embodiments of the methods, the attenuation of T cell activity is measured by increase of T cell exhaustion.

In some embodiments of the methods, the attenuation of T cell activity is measured by downregulation of T cell activation markers.

In some embodiments of the methods, the attenuation of T cell activity is measured by upregulation of regulatory T cell biomarkers.

In some embodiments of the methods, the attenuation of T cell activity is measured by increase of regulatory T cell numbers.

In certain embodiments of the methods, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof. In another embodiment, the downregulation occurs as early as 6 hours after the contact. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact. In still another embodiment, the downregulation occurs as early as 10 hours after the contact. In one embodiment, the downregulation occurs as early as 12 hours after the contact. In another embodiment, the downregulation occurs as early as 14 hours after the contact. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact. In still another embodiment, the downregulation occurs as early as 18 hours after the contact. In one embodiment, the downregulation occurs as early as 20 hours after the contact. In another embodiment, the downregulation occurs as early as 22 hours after the contact. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact. In some embodiments, the contact is with the antibody. In other embodiments, the contact is with an antigen-binding fragment thereof.

In some embodiments, the downregulation of PD-1 expression on the surface of T cells precedes cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 6 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In still another embodiment, the downregulation occurs as early as 10 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 12 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 14 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In still another embodiment, the downregulation occurs as early as 18 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 20 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 22 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition.

In other embodiments, the downregulation of PD-1 expression on the surface of T cells is concurrent with cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 6 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In still another embodiment, the downregulation occurs as early as 10 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 12 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 14 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In still another embodiment, the downregulation occurs as early as 18 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 20 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 22 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition.

In yet other embodiments, the downregulation of PD-1 expression on the surface of T cells is after cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 6 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In still another embodiment, the downregulation occurs as early as 10 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 12 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 14 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In still another embodiment, the downregulation occurs as early as 18 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 20 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 22 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition.

In one embodiment of the methods, the K_(D) of the antibody or antigen-binding fragment thereof for binding to purified human PD-1 is from about 1 nM to about 100 nM. In another embodiment of the methods, the K_(D) of the antibody or antigen-binding fragment thereof for binding to human PD-1 expressed on cell surface and monkey PD-1 expressed on cell surface is from about 100 pM to about 10 nM.

In some embodiments of the methods, the EC₅₀ of the antibody or antigen-binding fragment thereof for attenuating T cell activity is from about 1 pM to about 10 pM, from about 10 pM to about 100 pM, from about 100 pM to about 1 nM, from about 1 nM to about 10 nM, or from about 10 nM to about 100 nM.

In other embodiments of the methods, the maximal percent attenuation of T cell activity by the antibody or antigen-binding fragment thereof is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

In another embodiment of the methods, the maximal percent downregulation of PD-1 expression by the antibody or antigen-binding fragment thereof is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

In certain embodiments of the methods, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized, human, or chimeric antibody. In another embodiment, the humanized antibody is a deimmunized antibody or a composite human antibody. In certain embodiments, the antibody is a humanized antibody. In specific embodiments, the antibody is a humanized antibody that specifically binds human PD-1.

In certain embodiments of the methods, the antibody or antigen-binding fragment thereof is a Fab, a Fab′, a F(ab′)₂, a Fv, a scFv, a dsFv, a diabody, a triabody, or a tetrabody. In some embodiments, the antibody or antigen-binding fragment thereof is a multispecific antibody formed from antibody fragments. In other embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody.

In some embodiments of the methods, the antibody or antigen-binding fragment thereof is conjugated to an agent. In one embodiment, the agent is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof attenuates T cell activity. In one embodiment, the maximal percent attenuation of T cell activity is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the attenuation of T cell activity is measured by inhibition of T cell proliferation. In some embodiments, the attenuation of T cell activity is measured by inhibition of cytokine production. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-17, IFN-γ, or any combination thereof. In certain embodiments, the cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, IFN-γ, and TNF-α. In certain embodiments, the cytokine is IL-1. In some embodiments, the cytokine is 1-2. In other embodiments, the cytokine is IL-6. In another embodiment, the cytokine is 1-12. In some other embodiments, the cytokine is 1-17. In yet other embodiments, the cytokine is 1-22. In still other embodiments, the cytokine is IL-23. In some embodiments, the cytokine is GM-CSF. In other embodiments, the cytokine is IFN-γ. In yet other embodiments, the cytokine is TNF-α. In certain embodiments, the cytokine is IL-2 and 1-17. In some embodiments, the cytokine is IL-2 and IFN-γ. In yet other embodiments, the cytokine is IL-17 and IFN-γ. In still other embodiments, the cytokine is IL-2, IL-17, and IFN-γ. Other combinations of two, three or more of the above-mentioned cytokines are also contemplated. In certain embodiments, the inhibition of cytokine production follows downregulation of PD-1 expression on the surface of the T cell. In other embodiments, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after the contact with the antibody or antigen-binding fragment thereof. In one embodiment, the downregulation occurs as early as 4 hours after the contact, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 6 hours after the contact, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 8 hours after the contact, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 10 hours after the contact, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 12 hours after the contact, and precedes cytokine inhibition. In an embodiment, the downregulation occurs as early as 14 hours after the contact, and precedes cytokine inhibition. In other embodiments, the downregulation occurs as early as 16 hours after the contact, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 18 hours after the contact, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 20 hours after the contact, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 22 hours after the contact, and precedes cytokine inhibition. In some embodiments, the downregulation occurs as early as 24 hours after the contact, and precedes cytokine inhibition. In some embodiments, the inhibition of cytokine production is concurrent with downregulation of PD-1 expression on the surface of the T cell. In one embodiment, the downregulation occurs as early as 4 hours after the contact, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 6 hours after the contact, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 8 hours after the contact, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 10 hours after the contact, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 12 hours after the contact, and is concurrent with cytokine inhibition. In an embodiment, the downregulation occurs as early as 14 hours after the contact, and is concurrent with cytokine inhibition. In other embodiments, the downregulation occurs as early as 16 hours after the contact, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 18 hours after the contact, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 20 hours after the contact, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 22 hours after the contact, and is concurrent with cytokine inhibition. In some embodiments, the downregulation occurs as early as 24 hours after the contact, and is concurrent with cytokine inhibition. In some embodiments, the inhibition of cytokine production proceeds downregulation of PD-1 expression on the surface of the T cell. In one embodiment, the downregulation occurs as early as 4 hours after the contact, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 6 hours after the contact, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 8 hours after the contact, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 10 hours after the contact, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 12 hours after the contact, and is after cytokine inhibition. In an embodiment, the downregulation occurs as early as 14 hours after the contact, and is after cytokine inhibition. In other embodiments, the downregulation occurs as early as 16 hours after the contact, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 18 hours after the contact, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 20 hours after the contact, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 22 hours after the contact, and is after cytokine inhibition. In some embodiments, the downregulation occurs as early as 24 hours after the contact, and is after cytokine inhibition.

In yet another embodiment of the methods, the antibody or antigen-binding fragment thereof downregulates PD-1 expression on the surface of a T cell. In one embodiment, the maximal percent downregulation of PD-1 expression by the antibody or antigen-binding fragment thereof is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In another embodiment, the downregulation of PD-1 expression on the surface of the T cell occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof. In one embodiment, the downregulation of PD-1 expression on the surface of the T cell precedes cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of the T cell is concurrent with cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of the T cell follows cytokine inhibition. In certain embodiments, the cytokine is IL-2, IL-17, IFN-γ, or any combination thereof. In certain embodiments, the cytokine is selected from the group consisting of IL-1, 11-2, 11-6, 11-12, 11-17, IL-22, IL-23, GM-CSF, IFN-γ, and TNF-α. In certain embodiments, the cytokine is IL-1. In some embodiments, the cytokine is IL-2. In other embodiments, the cytokine is 11-6. In another embodiment, the cytokine is IL-12. In other embodiments, the cytokine is IL-17. In yet other embodiments, the cytokine is IL-22. In still other embodiments, the cytokine is IL-23. In some embodiments, the cytokine is GM-CSF. In other embodiments, the cytokine is IFN-γ. In yet other embodiments, the cytokine is TNF-α. In certain embodiments, the cytokine is IL-2 and 11-17. In some embodiments, the cytokine is IL-2 and IFN-γ. In yet other embodiments, the cytokine is 11-17 and IFN-γ. In still other embodiments, the cytokine is IL-2, IL-17, and IFN-γ.

In another embodiment of the methods, the antibody or antigen-binding fragment thereof attenuates T cell activity by inhibiting cytokine production. In some embodiments, the cytokine that is inhibited by the antibody or antigen-binding fragment thereof is selected from the group consisting of IL-2, 11-17, IFN-γ, or any combination thereof. In certain embodiments, the cytokine is selected from the group consisting of IL-1, 11-2, 11-6, 11-12, 11-17, 11-22, 11-23, GM-CSF, IFN-γ, and TNF-α. In certain embodiments, the cytokine is IL-1. In some embodiments, the cytokine is IL-2. In other embodiments, the cytokine is 11-6. In another embodiment, the cytokine is 11-12. In other embodiments, the cytokine is 11-17. In yet other embodiments, the cytokine is IL-22. In still other embodiments, the cytokine is IL-23. In some embodiments, the cytokine is GM-CSF. In other embodiments, the cytokine is IFN-γ. In yet other embodiments, the cytokine is TNF-α. In certain embodiments, the cytokine is IL-2 and IL-17. In some embodiments, the cytokine is IL-2 and IFN-γ. In yet other embodiments, the cytokine is 11-17 and IFN-γ. In still other embodiments, the cytokine is IL-2, 11-17, and IFN-γ. Other combinations of two, three or more of the above-mentioned cytokines are also contemplated.

Also provided herein is a method of managing, preventing, or treating an immune disorder in a subject, comprising administering to a subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein. In certain embodiments of the methods, the composition further comprises a pharmaceutically acceptable carrier.

The present disclosure provides a method of managing, preventing, or treating an immune disorder in a subject, comprising administering to a subject a therapeutically effective amount of any antibody or antigen-binding fragment thereof described herein. In some embodiments, the immune disorder is an inflammatory disease. In one embodiment, the inflammatory disease is uveitis. In other embodiments, the immune disorder is a hypersensitivity disease. In one embodiment, the hypersensitivity disease is a T cell hypersensitivity disease. In still other embodiments, the immune disorder is an autoimmune disease.

In certain embodiments, the hypersensitivity disease is allergy, atopic dermatitis, or hypersensitivity vasculitis. In one embodiment, the hypersensitivity disease is allergy. In other embodiments, the hypersensitivity disease is atopic dermatitis. In still other embodiments, the hypersensitivity disease is hypersensitivity vasculitis.

In certain embodiments, the immune disorder is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, multiple sclerosis, lupus, ankylosing spondylitis, type I diabetes, Sjögren's syndrome, ulcerative colitis, neuromyelitis optica, celiac disease, scleroderma, and temporal arteritis. In certain embodiments, the lupus is systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), or lupus nephritis. In one embodiment, the autoimmune disease is rheumatoid arthritis. In another embodiment, the autoimmune disease is Crohn's disease. In yet another embodiment, the autoimmune disease is psoriasis. In still another embodiment, the autoimmune disease is multiple sclerosis. In another embodiment, the autoimmune disease is ankylosing spondylitis. In yet another embodiment, the autoimmune disease is type I diabetes. In still another embodiment, the autoimmune disease is Sjögren's syndrome. In a further embodiment, the autoimmune disease is ulcerative colitis. In another embodiment, the autoimmune disease is neuromyelitis optica. In yet another embodiment, the autoimmune disease is celiac disease. In one embodiment, the autoimmune disease is temporal arteritis. In another embodiment, the autoimmune disease is scleroderma. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is SLE. In another embodiment, the autoimmune disease is CLE. In still another embodiment, the autoimmune disease is lupus nephritis.

In certain embodiments, the subject has the autoimmune disease, or is being treated for the autoimmune disease with an anti-inflammatory therapeutic.

In certain other embodiments, the immune cells in the subject express PD-1.

3. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show that the T cell attenuating anti-PD-1 antibodies (PD1AB) do not compete with PD-L1 (PD-L-DyL650 denotes PD-L1 conjugated with the dye DyL650) binding to PD-1: (A) PD1AB-1, PD1AB-2, and PD1AB-6; (B) PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, and PD1AB-5. MDX 4H1, an antagonist antibody, blocks PD-L1 binding to PD-1.

FIG. 2 depicts that the PD-1:PD1AB-6 Fab interaction site is at a distal side of PD-1 relative to the PD-1:PD-L1 interaction site.

FIG. 3 depicts that PD1AB-6 Fab binds against a PD-1 β sheet, with substantial interactions formed with a PD-1 loop composed of residues 100-105.

FIG. 4 shows the amino acid sequences of heavy chain (HC) and light chain (LC) of PD1AB-6-IgG1 and HC of its variants PD1AB-6-K3 and PD1AB-6-4P.

FIGS. 5A-5B depict the PD1AB-6-IgG1 affinity for cyno (A) or human (B) PD-1 expressed on CHO cells.

FIG. 6 depicts the binding of PD1AB-6-IgG1, isotype control, and human PD-L1 Fc fusion protein (hPD-L1 Fc) to activated human PBMC gated on CD4+ T cells.

FIG. 7 depicts the binding of PD1AB-6-IgG1, isotype control, and human PD-L1 Fc fusion protein (hPD-L1 Fc) to activated cyno PBMC gated on CD4+ T cells.

FIGS. 8A-8D show the PD1AB-6 variants binding to FcγRI (A), FcγRIIIa (V158) (B), or FcγRIIb (C) expressed on HEK293 cells using Cisbio Tag-Lite™ detection, and (D) the EC₅₀ values of the PD1AB-6 variants binding to FcγRI, FcγRIIIa (V158), or FcγRIIb.

FIGS. 9A-9C depict the PD1AB-6 variants binding to FcγRIIIa (V158) (A) or FcγRI (B) expressed on CHO cells using FACS, and (C) the EC₅₀ values of the PD-1 antibody variants binding to FcγRI or FcγRIIIa.

FIGS. 10A-10B depict the ADCC activity of the PD1AB-6 variants and a control human IgG1 Fc among two representatives of four individual healthy donors: (A) Donor 7 and (B) Donor 8.

FIG. 11 depicts the CDC activity of the PD1AB-6 variants. Data are representative of 3 independent experiments: (i) CDC activity of PD1AB-6-IgG1 and anti-human CD20 IgG; (ii) CDC activity of PD1AB-6-IgG1 and PD1AB-6-K3; (iii) CDC activity of PD1AB-6-4P and commercial human IgG4 isotype control antibody and human IgG1 Fc protein.

FIG. 12 depicts the potent attenuating activity of PD1AB-6 variants in human PBMC assay, measured by I1-2 levels in culture supernatants at 24 hours post-stimulation.

FIG. 13 depicts the activity of PD1AB-6-K3 in human whole blood assay. The graph shows a representative curve from donor 4 used to calculate EC₅₀ of IFN-γ inhibition. The table shows EC₅₀ values of IFN-γ inhibition for 4 healthy donors with PD1AB-6 variants and CTLA4Ig.

FIGS. 14A-14C depict downregulation of PD-1 expression by PD1AB-6-IgG1 as determined by (A) isotype vs. PD-1 staining on CD3+ T cells in human PBMC activated with anti-CD3+anti-CD28 for 48 hours, (B) PD-1 expression in isotype IgG vs. PD1AB-6-IgG1 treated PBMC (the detection anti-PD-1 antibody is not blocked by PD1AB-6), and (C) PD-1 expression on CD3+ T cells in human PBMC from 3 different donors, activated with anti-CD3+anti-CD28 and three different concentrations of either isotype IgG or PD1AB-6-IgG.

FIGS. 15A-15C show (A) PD AB-6-IgG1, (B) PD1AB-6-4P, and (C) PD1AB-6-K3 binding to PD-1 antigen on Biacore® T200.

FIG. 16 shows that PD1AB-6-IgG1 inhibited CD4+ T cell tetanus recall response with similar IC₅₀ values in PBMCs from three different donors.

FIGS. 17A-17B show that PD1AB-6-K3 blocked secretion of IFN-γ (A) and IL-17 (B) in whole blood samples from autoimmune disease patients.

FIG. 18 shows that PD1AB-6-K3 reduced unexhausted IL-7R^(hi) CD8+ T cells.

FIGS. 19A-19B show that PD1AB-6-K3 inhibited proliferation of (A) isolated CD4+ T cells and (B) isolated CD8+ T cells.

FIG. 20 shows that PD1AB-6-IgG1 increased CD25+Foxp3+ induced regulatory T cells at various concentrations of transforming growth factor beta (TGF-β).

FIG. 21 shows strong PD-1 expression in lymphocytes in follicular formation (left panel) and low PD-L1 expression in lymphoid aggregates (right panel) in synovium of RA patient 5.

FIG. 22 shows rare PD-1+ lymphocytes in lymphoid aggregate in connective tissue in joint (left panel) and rare PD-L1+ antigen-presenting cells in similar location (right panel) of RA patient 1.

FIG. 23 shows that PD-1+ cells predominate in follicles and are rare in the deep lamina propria (left panel), and that PD-L1 are widely expressed in follicles and in macrophages and lymphocytes in lamina propria (right panel) in the intestines of Crohn's patient.

FIG. 24 shows rare PD-1+ lymphocytes along lamina propria near ulcerated region (left panel) and numerous PD-L1+ macrophages in the same region (right panel) of Crohn's disease intestinal sample (40X).

FIGS. 25A-25C show PD-1 and PD-L1 expression scores in pilot study of (A) Crohn's, (B) Rheumatoid Arthritis (RA), and (C) psoriasis.

FIGS. 26A-26B show that PD1AB-6-K3, with reduced Clq binding, has similar inhibitory activity as PD1AB-6-IgG1 molecule in PBMC transfer xeno-GvHD model, as determined by (A) hGM-CSF levels and (B) number of human CD45+ cells in the spleen.

FIGS. 27A-27B show that PD1AB-6-K3 inhibited human CD45+ lymphocyte engraftment in blood (A) and spleen (B) in CD4+ T cell transfer xeno-GvHD model.

FIGS. 28A-28C show that PD1AB-6-K3 inhibited secretion of GM-CSF (A), TNF-α (B), and 11-17 (C) in CD4+ T cell transfer xeno-GvHD model.

FIGS. 29A-29B show that PD1AB-6-K3 induced downregulation of PD-1 expression (A) precedes inhibition of cytokine GM-CSF secretion (B).

FIGS. 30A-30B show correlation between PD1AB-6 variants-induced downregulation of PD-1 expression and inhibition of cytokine secretion, exemplified by IL-17 inhibition (A) and IFN-γ inhibition (B).

FIG. 31 shows that a murine surrogate of PD1AB-6-K3 inhibited disease score in an experimental autoimmune encephalomyelitis (EAE) model.

FIGS. 32A-32B show that a murine surrogate of PD1AB-6-K3 inhibited IFN-γ secretion (A) and IL-17 secretion (B) in a recall assay with spleens on day 23 in the EAE model.

FIG. 33 shows differentiation of CD20+CD38-activated B-lymphocytes into CD20-CD38+ plasmablasts in in vitro T cell/B cell co-culture system using purified CD3+ T cells and total B cells from four exemplary donors.

FIGS. 34A-34C show percentage of reduction of plasmablast numbers (A), IgG secretion (B), and IgM surface expression (C) induced by PD1AB-6-K3 in T cell receptor activated CD3+ T Cell/total B Cell co-cultures.

FIGS. 35A-35C show percentage of reduction of plasmablast numbers (A), IgG secretion (B), and IgM surface expression (C) induced by PD-L1 Fc fusion protein in T cell receptor activated CD3+ T Cell/total B Cell co-cultures.

4. DETAILED DESCRIPTION

Provided herein are methods of managing, preventing, or treating an immune disorder in a subject, comprising administering to a subject an effective amount of binding proteins, such as antibodies that bind to PD-1. In one embodiment, provided herein is a method of managing an immune disorder in a subject, comprising administering to a subject an effective amount of a PD-1 binding protein. In one embodiment, provided herein is a method of preventing an immune disorder in a subject, comprising administering to a subject an effective amount of a PD-1 binding protein. In one embodiment, provided herein is a method of treating an immune disorder in a subject, comprising administering to a subject an effective amount of a PD-1 binding protein. In a specific embodiment, the PD-1 binding protein is an antibody that binds to PD-1. Exemplary PD-1 antibodies useful in these methods are provided herein.

In some embodiments of the various methods provided herein, the antibodies bind to human and/or cyno PD-1. In some embodiments, the binding proteins, such as antibodies that bind to human and/or cyno PD-1, do not bind to rodent PD-1. In certain embodiments, the PD-1 binding proteins, including antibodies disclosed herein, are agonists (e.g., can mimic the effect of PD-1 ligand and induce PD-1 signaling). In some embodiments, the binding proteins such as antibodies to PD-1 provided herein (i) bind to human and/or cyno PD-1, (ii) do not compete for binding with PD-1 ligand (e.g., PD-L1 and/or PD-L2), and/or (iii) induce PD-1 signaling. In one embodiment, the PD-1 antibodies bind to human PD-1. In one embodiment, the PD-1 antibodies bind to cyno PD-1. In one embodiment, the PD-1 antibodies bind to both human PD-1 and cyno PD-1. In some embodiments, the PD-1 antibodies do not compete with PD-L1 for binding to PD-1. In other embodiments, the PD-1 antibodies do not compete with PD-L2 for binding to PD-1. In yet other embodiments, the PD-1 antibodies do not compete with either PD-L1 or PD-L2 for binding to PD-1. In other embodiments, the PD-1 antibodies induce PD-1 signaling. In specific embodiments, the PD-1 antibodies provided herein bind to both human PD-1 and cyno PD-1, do not compete for binding to PD-1 with either PD-L1 or PD-L2, and induce PD-1 signaling. In some embodiments, the binding, competition, and/or signaling is assayed in vitro, e.g., in a cell-based assay. In other embodiments, the binding, competition, and/or signaling is assayed ex vivo, e.g., in a T cell function assay. In other embodiments, the binding, competition, and/or signaling is assayed using a sample from a subject (e.g., a human subject). In certain embodiments, assays and measurements include (1) a human or cyno PBMC assay (see, e.g., Examples 5.2.1 and 5.2.2); (2) a human whole blood sample assay (see, e.g., Examples 5.2.1 and 5.4.2); (3) a Tetanus Toxoid Antigen (TTX) recall assay (see, e.g., Example 5.4.1); (4) measuring expression of a T cell inhibitory receptor (see, e.g., Example 5.4.3 and 5.4.6); (5) measuring T cell proliferation (see, e.g., Example 5.4.4); and (6) measuring numbers of regulatory T cells (see, e.g., Example 5.4.5). In certain embodiments, binding proteins, such as anti-PD-1 antibodies, as described herein, exhibit activities that are consistent with the natural biological function of PD-L1 and/or PD-L2. In some embodiments, the activities are exhibited in vitro. In other embodiments, the activities are exhibited ex vivo.

In other embodiments, the binding, competition and/or signaling is assayed in vivo, e.g., in a xeno-graft versus host disease (xeno-GvHD) model for human diseases. In specific embodiments, the binding, competition and/or signaling is assayed using an animal disease model (e.g., a mouse disease model). In specific embodiments, the binding, competition and/or signaling is assayed using a sample from a disease model subject (e.g., a mouse disease model subject). In certain embodiments, animal disease models and assays include (1) a xeno-GvHD model (see, e.g., Example 5.5.1); and (2) an experimental autoimmune encephalomyelitis (EAE) model in human PD-1 knock-in (KI) mice (see, e.g., Example 5.5.2). In certain embodiments, binding proteins, such as anti-PD-1 antibodies, as described herein, exhibit activities that are consistent with the natural biological function of PD-L1 and/or PD-L2. In other embodiments, the activities are exhibited in vivo.

The discovery that such binding proteins, including anti-PD-1 antibodies, induce PD-1 signaling make them viable therapeutics for the treatment of immune disorders. In certain embodiments, the immune disorder is an inflammatory disease. In one embodiment, the inflammatory disease is uveitis. In other embodiments, the immune disorder is a hypersensitivity disease. In some embodiments, the hypersensitivity disease is a T cell hypersensitivity disease. In certain embodiments, the hypersensitivity disease is allergy. In other embodiments, the hypersensitivity disease is atopic dermatitis. In yet other embodiments, the hypersensitivity disease is hypersensitivity vasculitis. In some embodiments, the immune disorders is an autoimmune disease. In one embodiment, the autoimmune disease is rheumatoid arthritis. In another embodiment, the autoimmune disease is Crohn's disease. In yet another embodiment, the autoimmune disease is psoriasis. In still another embodiment, the autoimmune disease is multiple sclerosis. In another embodiment, the autoimmune disease is ankylosing spondylitis. In yet another embodiment, the autoimmune disease is type I diabetes. In still another embodiment, the autoimmune disease is Sjögren's syndrome. In a further embodiment, the autoimmune disease is ulcerative colitis. In another embodiment, the autoimmune disease is neuromyelitis optica. In yet another embodiment, the autoimmune disease is celiac disease. In one embodiment, the autoimmune disease is temporal arteritis. In another embodiment, the autoimmune disease is scleroderma. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is SLE. In another embodiment, the autoimmune disease is CLE. In still another embodiment, the autoimmune disease is lupus nephritis. This discovery can also make the disclosed binding proteins, including anti-PD-1 antibodies, viable therapeutics broadly for the treatment of any disease, disorder, or condition in which it is desirable to mimic or augment PD-1 signaling (e.g., in vivo or in vitro).

In specific embodiments of the various methods provided herein, the binding proteins, such as antibodies that bind to PD-1, described herein share the common feature of competing with each other for the binding of PD-1. This competitive inhibition can indicate that each antibody binds to the same region of PD-1 (e.g., the same epitope), thereby asserting similar effects. In certain embodiments, anti-PD-1 antibodies provided herein include humanized anti-PD-1 antibodies, such as those derived from or based on antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and/or PD1AB-6. In other embodiments, anti-PD-1 antibodies provided herein compete for binding with an antibody derived from or based on PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and/or PD1AB-6. In some embodiments, the anti-PD-1 antibodies have CDR sequences as described in Tables 1-2. In certain embodiments, the anti-PD-1 antibodies bind to a specific domain or epitope of human PD-1 (e.g., residues 100-105; see Example 5.1.4). Moreover, such binding can be largely attributed to particular amino acid residues within the region (e.g., G103 and R104; see Example 5.1.4), which comprise the epitope recognized by the anti-PD-1 antibodies provided herein. Taken together, the results described herein demonstrate that the effects observed for an anti-PD-1 antibody that is derived from or based on PD1AB-6, including an antibody having one or more CDRs described in Tables 1-2, can be extrapolated to other anti-PD-1 antibodies provided herein having the same or similar epitope specificity (e.g., the same or similar CDRs). For example, the activities of antibodies as shown in Examples 5.1.2-3, 5.1.7-10, 5.2.1-3, 5.3.1, 5.4.1-4, 5.5.1-3, for an exemplary humanized anti-PD-1 antibody, are representative of the activities and effects of the anti-PD-1 antibodies provided herein.

In some embodiments of the various methods provided herein, the binding proteins such as anti-PD-1 antibodies may comprise immunoglobulin variable regions which comprise one or more CDRs as described in Tables 1-2. In such binding proteins (e.g., anti-PD-1 antibodies), the CDRs may be joined with one or more scaffold regions or framework regions (FRs), which orient(s) the CDR(s) such that the proper antigen binding properties of the CDR(s) is achieved. Such binding proteins, including anti-PD-1 antibodies as described herein, can induce PD-1 signaling.

4.1 General Techniques

Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dübel eds., 2d ed. 2010).

4.2 Terminology

Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

The terms “Programmed Death 1,” “Programmed Cell Death 1,” “Protein PD-1,” “PD-1,” “PD-1 polypeptide,” or “PD1” encompasses a polypeptide (“polypeptide” and “protein” are used interchangeably herein), including any native polypeptide, from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. In certain embodiments, the terms include “related PD-1 polypeptides,” including SNP variants thereof. The term “PD-1” also encompasses “full-length,” unprocessed PD-1 as well as any form of PD-1 that results from processing in the cell. In some embodiments, the PD1 has an amino acid sequence of SEQ ID NO:43. GenBank™ accession number U64863 provides another exemplary human PD-1 nucleic acid sequence.

“Related PD-1 polypeptides” include allelic variants (e.g., SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, which can retain PD-1 activity. As those skilled in the art will appreciate, an anti-PD-1 antibody provided herein can bind to a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 antigen, and/or a PD-1 epitope. An “epitope” may be part of a larger PD-1 antigen, which may be part of a larger PD-1 polypeptide fragment, which, in turn, may be part of a larger PD-1 polypeptide. PD-1 may exist in a native or denatured form. PD-1 polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Orthologs to the PD-1 polypeptide are also well known in the art.

The terms “PD AB-6-IgG1,” “PD AB-6 IgG,” “PD AB-6_IgG1,” “IgG1_PD1AB-6,” and “IgG1-PD1AB-6” are used interchangeably, and refer to the antibody PD1AB-6 having an IgG1 Fc region. In certain embodiments, the antibody PD1AB-6 comprises a light chain amino acid sequence of LC_PD1AB-6-IgG1 (SEQ ID NO:31) and a heavy chain amino acid sequence of HC_PD1AB-6-IgG1 (SEQ ID NO:32), e.g., as shown in FIG. 4.

The terms “PD1AB-6-K3,” “PD1 AB-6-IgG1-K322A,” “PD1 AB-6-K322A,” “IgG1_PD1AB-6_K322A,” “IgG1_PD1AB-6_K3,” “IgG1-PD1AB-6-K322A,” and “IgG1-PD1AB-6-K3” are used interchangeably and refer to the PD1AB-6 variant having a K322A substitution in the IgG1 Fc region. In certain embodiments, the PD1AB-6 variant has a heavy chain amino acid sequence of HC_PD1AB-6-IgG1-K322A (SEQ ID NO:33), e.g., as shown in FIG. 4.

The terms “PD1AB-6-4P,” “IgG4P_PD1AB-6,” “IgG4P-PD1AB-6,” “PD AB-6_IgG4P,” and “PD AB-6-IgG4P” are used interchangeably and refer to the PD1AB-6 variant having an IgG4P Fc region. In certain embodiments, the PD-1 antibody variant has a heavy chain amino acid sequence of HC_PD1AB-6-IgG4P (SEQ ID NO:34), e.g., as shown in FIG. 4.

The terms “PD1AB-6-4PE,” “IgG4PE_PD1AB-6,” “IgG4PE-PD1AB-6,” and “PD1AB-6_IgG4PE,” and “PD1AB-6-IgG4PE” are used interchangeably and refer to the PD1AB-6 variant having an IgG4PE heavy chain amino acid sequence as HC_PD1AB-6-IgG4PE (SEQ ID NO:35).

The term “PD-1 ligand” refers to a molecule that binds to PD-1, e.g., in vivo or in vitro. Non-limiting examples of PD-1 ligand include naturally occurring ligands, e.g., PD-1 ligand 1 (PD-L1, also known as B7-H1 or CD274) and PD-1 ligand 2 (PD-L2, also known as B7-DC or CD273), and artificially generated ligands.

The terms “PD-L1” and “PDL-1” are used interchangeably herein and refer to PD-1 ligand 1 (also known as B7-H1 or CD274).

The terms “PD-1 activity,” “PD-1 signaling,” and “PD-1 ligand-like signaling” when applied to a binding protein such as an antibody that binds to PD-1 of the present disclosure, means that the binding protein (e.g., antibody) mimics or modulates a biological effect induced by the binding of PD-1 ligand, and induces a biological response that otherwise would result from PD-1 ligand binding to PD-1, e.g., in vivo or in vitro. In assessing the binding specificity of anti-PD-1 antibody, for example, an antibody or fragment thereof that binds to PD-1 (e.g., human PD-1), the antibody is deemed to induce a biological response when the response is equal to or greater than 5%, such as equal to or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% of the activity of a wild type PD-1 ligand standard. In one embodiment, the anti-PD-1 antibody or the PD-1 ligand is immobilized (for example, on a plastic surface or bead). In certain embodiments, the antibody has the following properties: exhibits an efficacy level of equal to or more than 5% of a PD-1 ligand standard, with an EC₅₀ of equal to or less than 100 nM, e.g., 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, or 0.1 nM in, for example, (1) a human or cyno PBMC assay (see, e.g., Examples 5.2.1 and 5.2.2); (2) a human whole blood sample assay (see, e.g., Examples 5.2.1 and 5.4.2); (3) a TTX recall assay (see, e.g., Example 5.4.1); (4) a T cell inhibitory receptor expression assay (see, e.g., Example 5.4.3 and 5.4.6); (5) a T cell proliferation assay (see, e.g., Example 5.4.4); (6) a regulatory T cells assay (see, e.g., Example 5.4.5); (7) a xeno-GvHD model (see, e.g., Example 5.5.1); and (8) an EAE model in human PD-1 KI mice (see, e.g., Example 5.5.2).

The term “binding protein” refers to a protein comprising a portion (e.g., one or more binding regions such as CDRs) that binds to PD-1, including human and/or cyno PD-1 and, optionally, a scaffold or framework portion (e.g., one or more scaffold or framework regions) that allows the binding portion to adopt a conformation that promotes binding of the binding protein to a PD-1 polypeptide, fragment, or epitope. Examples of such binding proteins include antibodies, such as a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab′)₂ fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, and fragments thereof. The binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, e.g., Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics 53(1): 121-29; and Roque et al., 2004, Biotechnol. Prog. 20:639-54. In addition, peptide antibody mimetics (“PAMs”) can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold. In the context of the present disclosure, a binding protein is said to specifically bind or selectively bind to PD-1, for example, when the dissociation constant (K_(D)) is ≦10⁻⁷ M. In some embodiments, the binding proteins (e.g., antibodies) may specifically bind to PD-1 with a K_(D) of from about 10⁻⁷ M to about 10⁻¹² M. In certain embodiments, the binding protein (e.g., antibody) may specifically bind to PD-1 with high affinity when the K_(D) is ≦10⁻⁸M or K_(D) is ≦10⁻⁹M. In one embodiment, the binding proteins (e.g., antibodies) may specifically bind to purified human PD-1 with a K_(D) of from 1×10⁻⁹M to 10×10⁻⁹M as measured by Biacore®. In another embodiment, the binding proteins (e.g., antibodies) may specifically bind to purified human PD-1 with a K_(D) of from 0.1×10⁻⁹M to 1×10⁻⁹M as measured by KinExA™ (Sapidyne, Boise, Id.). In yet another embodiment, the binding proteins (e.g., antibodies) specifically bind to human PD-1 expressed on cells with a K_(D) of from 0.1×10⁻⁹M to 10×10⁻⁹M. In certain embodiments, the binding proteins (e.g., antibodies) specifically bind to human PD-1 expressed on cells with a K_(D) of from 0.1×10⁻⁹ M to 1×10⁻⁹M. In some embodiments, the binding proteins (e.g., antibodies) specifically bind to human PD-1 expressed on cells with a K_(D) of 1×10⁻⁹M to 10×10⁻⁹M. In certain embodiments, the binding proteins (e.g., antibodies) specifically bind to human PD-1 expressed on cells with a K_(D) of about 0.1×10⁻⁹M, about 0.5×10⁻⁹ M, about 1×10⁻⁹M, about 5×10⁻⁹M, about 10×10⁻⁹ M, or any range or interval thereof. In still another embodiment, the binding proteins (e.g., antibodies) may specifically bind to cyno PD-1 expressed on cells with a K_(D) of 0.1×10⁻⁹M to 10×10⁻⁹M. In certain embodiments, the binding proteins (e.g., antibodies) specifically bind to cyno PD-1 expressed on cells with a K_(D) of from 0.1×10⁻⁹ M to 1×10⁻⁹M. In some embodiments, the binding proteins (e.g., antibodies) specifically bind to cyno PD-1 expressed on cells with a K_(D) of 1×10⁻⁹M to 10×10⁻⁹M. In certain embodiments, the binding proteins (e.g., antibodies) specifically bind to cyno PD-1 expressed on cells with a K_(D) of about 0.1×10⁻⁹ M, about 0.5×10⁻⁹M, about 1×10⁻⁹M, about 5×10⁻⁹M, about 10×10⁻⁹ M, or any range or interval thereof.

The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, individual anti-PD-1 monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), anti-PD-1 antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain anti-PD-1 antibodies, and fragments of anti-PD-1 antibodies, as provided below. An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). In specific embodiments, the specific molecular antigen can be bound by an antibody provided herein, including a PD-1 polypeptide, a PD-1 fragment, or a PD-1 epitope. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments such as PD-1-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments such as PD-1-binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab′) fragments, F(ab)₂ fragments, F(ab′)₂ fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to a PD-1 antigen (e.g., one or more CDRs of an anti-PD-1 antibody). Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. Anti-PD-1 antibodies may be agonistic antibodies or antagonistic antibodies. Provided herein are agonistic antibodies to PD-1, including antibodies that induce PD-1 signaling. In specific embodiments, agonistic antibodies to PD-1 do not compete for the binding of PD-L1 and/or PD-L2 to PD-1.

An “antigen” is a predetermined antigen to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.

The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to that portion of an antibody, which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the CDRs).

The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as PD-1, is the affinity of the antibody or functional fragment for that epitope. The ratio of dissociation rate (k_(off)) to association rate (k_(on)) of an antibody to a monovalent antigen (k_(off)/k_(on)) is the dissociation constant K_(D), which is inversely related to affinity. The lower the K_(D) value, the higher the affinity of the antibody. The value of K_(D) varies for different complexes of antibody and antigen and depends on both k_(on) and k_(off). The dissociation constant K_(D) for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent PD-1, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.

The terms “antibodies that specifically bind to PD-1,” “antibodies that specifically bind to a PD-1 epitope,” and analogous terms are also used interchangeably herein and refer to antibodies that specifically bind to a PD-1 polypeptide, such as a PD-1 antigen, or fragment, or epitope (e.g., human PD-1 such as a human PD-1 polypeptide, antigen, or epitope). An antibody that specifically binds to PD-1 (e.g., human PD-1) may bind to the extracellular domain or peptide derived from the extracellular domain of PD-1. An antibody that specifically binds to a PD-1 antigen (e.g., human PD-1) may be cross-reactive with related antigens (e.g., cyno PD-1). In certain embodiments, an antibody that specifically binds to a PD-1 antigen does not cross-react with other antigens. An antibody that specifically binds to a PD-1 antigen can be identified, for example, by immunoassays, Biacore®, or other techniques known to those of skill in the art. An antibody binds specifically to a PD-1 antigen when it binds to a PD-1 antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs). Typically a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding antibody specificity. An antibody which “binds an antigen of interest” (e.g., a target antigen such as PD-1) is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a “non-target” protein will be less than about 10% of the binding of the antibody to its particular target protein, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. With regard to the binding of an antibody to a target molecule, the term “specific binding,” “specifically binds to,” or “is specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “anti-PD-1 antibody” or “an antibody that binds to PD-1” includes an antibody that is capable of binding PD-1 with sufficient affinity such that the antibody is useful, for example, as a diagnostic agent in targeting PD-1. The term “specific binding,” “specifically binds to,” or “is specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In certain embodiments, an antibody that binds to PD-1 has a dissociation constant (K_(D)) of less than or equal to 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, anti-PD-1 antibody binds to an epitope of PD-1 that is conserved among PD-1 from different species (e.g., between human and cyno PD-1).

The term “compete” when used in the context of anti-PD-1 antibodies (e.g., agonistic antibodies and binding proteins that bind to PD-1 and compete for the same epitope or binding site on a target) means competition as determined by an assay in which the antibody (or binding fragment) thereof under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand or reference antigen-binding protein, such as a reference antibody) to a common antigen (e.g., PD-1 or a fragment thereof). Numerous types of competitive binding assays can be used to determine if a test antibody competes with a reference antibody for binding to PD-1 (e.g., human PD-1). Examples of assays that can be employed include solid phase direct or indirect RIA, solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-53), solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-19), solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988)), solid phase direct label RIA using I-125 label (see, e.g., Morel et al., 1988, Mol. Immunol. 25:7-15), and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., PD-1 such as human PD-1) bound to a solid surface, or cells bearing either of an unlabelled test antigen-binding protein (e.g., test anti-PD-1 antibody) or a labeled reference antigen-binding protein (e.g., reference anti-PD-1 antibody). Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Usually the test antigen-binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and/or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur. Additional details regarding methods for determining competitive binding are described herein. Usually, when a competing antibody protein is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 30%, for example 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

An “isolated” antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source and/or other contaminant components from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”). In certain embodiments, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1% of the volume of the protein preparation. In certain embodiments, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, for example, it is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. Accordingly such preparations of the antibody have less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminant components can also include, but are not limited to, materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method (Lowry et al., 1951, J. Bio. Chem. 193: 265-75), such as 96%, 97%, 98%, or 99%, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In specific embodiments, antibodies provided herein are isolated.

A 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for pt and e isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994).

The term “variable region,” “variable domain,” “V region,” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH.” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a 3 sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the J sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. In specific embodiments, the variable region is a human variable region.

The term “variable region residue numbering as in Kabat” or “amino acid position numbering as in Kabat”, and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

An “intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2 and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. In certain embodiments, an intact antibody has one or more effector functions.

“Antibody fragments” comprise a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include, without limitation, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2003, Nat. Med. 9:129-34; WO 93/11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (sdAbs) (see, e.g., Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49); and multispecific antibodies formed from antibody fragments.

A “functional fragment,” “binding fragment,” or “antigen-binding fragment” of a therapeutic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least binding to the target antigen (e.g., a PD-1 binding fragment or fragment that binds to PD-1).

The term “fusion protein” as used herein refers to a polypeptide that comprises an amino acid sequence of an antibody and an amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein not normally a part of the antibody (e.g., a non-anti-PD-1 antigen-binding antibody)). The term “fusion” when used in relation to PD-1 or to an anti-PD-1 antibody refers to the joining of a peptide or polypeptide, or fragment, variant, and/or derivative thereof, with a heterologous peptide or polypeptide. In certain embodiments, the fusion protein retains the biological activity of the PD-1 or anti-PD-1 antibody. In certain embodiments, the fusion protein comprises a PD-1 antibody VH region, VL region, VH CDR (one, two, or three VH CDRs), and/or VL CDR (one, two, or three VL CDRs), wherein the fusion protein binds to a PD-1 epitope, a PD-1 fragment, and/or a PD-1 polypeptide.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) or lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “host” as used herein refers to an animal, such as a mammal (e.g., a human).

The term “host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen. In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single hybridoma or other cell, wherein the antibody binds to only a PD-1 epitope as determined, for example, by ELISA or other antigen-binding or competitive binding assay known in the art. The term “monoclonal” is not limited to any particular method for making the antibody. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., 1975, Nature 256:495, or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97, for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002). Exemplary methods of producing monoclonal antibodies are provided in the Examples herein.

The term “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not manipulated, modified, and/or changed (e.g., isolated, purified, selected) by a human being.

The antibodies provided herein can include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55).

“Humanized” forms of nonhuman (e.g., murine) antibodies are chimeric antibodies that include human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; U.S. Pat. Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

A “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1: 755-68). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5: 368-74. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62 regarding human antibodies generated via a human B-cell hybridoma technology.

A “CDR” refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., 1997, J. Biol. Chem. 252:6609-16; Kabat, 1978, Adv. Prot. Chem. 32:1-75). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact, and IMGT. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those skilled in the art.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dübel eds., 2d ed. 2010)). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.

Recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Plückthun, 2001, J. Mol. Biol. 309: 657-70. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). In some embodiments, the CDRs are as defined by the IMGT numbering system. In other embodiments, the CDRs are as defined by the Kabat numbering system. In certain embodiments, the CDRs are as defined by the AbM numbering system. In other embodiments, the CDRs are as defined by the Chothia system. In yet other embodiments, the CDRs are as defined by the Contact numbering system.

IMGT Kabat AbM Chothia Contact V_(H) CDR1 27-38 31-35 26-35 26-32 30-35 V_(H) CDR2 56-65 50-65 50-58 53-55 47-58 V_(H) CDR3 105-117  95-102  95-102  96-101  93-101 V_(L) CDR1 27-38 24-34 24-34 26-32 30-36 V_(L) CDR2 56-65 50-56 50-56 50-52 46-55 V_(L) CDR3 105-117 89-97 89-97 91-96 89-96

Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. As used herein, the terms “HVR” and “CDR” are used interchangeably.

The term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

The term “framework” or “FR” refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

An “affinity matured” antibody is one with one or more alterations (e.g., amino acid sequence variations, including changes, additions, and/or deletions) in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Affinity matured antibodies can have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For review, see Hudson and Souriau, 2003, Nature Medicine 9:129-34; Hoogenboom, 2005, Nature Biotechnol. 23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51.

A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. For example, blocking antibodies or antagonist antibodies may substantially or completely inhibit the biological activity of the antigen.

An “agonist” antibody is an antibody that triggers a response, e.g., one that mimics at least one of the functional activities of a polypeptide of interest (e.g., PD-L). An agonist antibody includes an antibody that is a ligand mimetic, for example, wherein a ligand binds to a cell surface receptor and the binding induces cell signaling or activities via an intercellular cell signaling pathway and wherein the antibody induces a similar cell signaling or activation. An “agonist” of PD-1 refers to a molecule that is capable of activating or otherwise increasing one or more of the biological activities of PD-1, such as in a cell expressing PD-1. In some embodiments, an agonist of PD-1 (e.g., an agonistic antibody as described herein) may, for example, act by activating or otherwise increasing the activation and/or cell signaling pathways of a cell expressing a PD-1 protein, thereby increasing a PD-1-mediated biological activity of the cell relative to the PD-1-mediated biological activity in the absence of agonist. In some embodiments the antibodies provided herein are agonistic anti-PD-1 antibodies, including antibodies that induce PD-1 signaling.

“Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (K_(D)). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In one embodiment, the “K_(D)” or “K_(D) value” may be measured by assays known in the art, for example by a binding assay. The K_(D) may be measured in a RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). The K_(D) or K_(D) value may also be measured by using surface plasmon resonance assays by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000, or by biolayer interferometry using, for example, the Octet®QK384 system. An “on-rate” or “rate of association” or “association rate” or “k_(on)” may also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a Biacore®TM-2000 or a Biacore®TM-3000, or the Octet®QK384 system.

The phrase “substantially similar” or “substantially the same” denotes a sufficiently high degree of similarity between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by the values (e.g., K_(D) values). For example, the difference between the two values may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5%, as a function of the value for the reference antibody.

The phrase “substantially increased,” “substantially reduced,” or “substantially different,” as used herein, denotes a sufficiently high degree of difference between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by the values. For example, the difference between said two values can be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50%, as a function of the value for the reference antibody.

The term “inhibition” or “inhibit,” when used herein, refers to partial (such as, 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (i.e., 100%) inhibition.

“Antibody effector functions” refer to the biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include but are not limited to: Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

“T cell effector functions” refer to the biological activities attributable to various types of T cells, including but not limited to cytotoxic T cells, T helper cells, regulatory T cells, and memory T cells. Examples of T cell effector functions include: increasing T cell proliferation, secreting cytokines, releasing cytotoxins, expressing membrane-associated molecules, killing target cells, activating macrophages, and activating B cells.

“T cell exhaustion” refers to a state of T cell dysfunction that arises during chronic infections, cancer, and other diseases, disorders, or conditions. The T cell exhaustion includes, but is not limited to, poor T cell effector functions (e.g., reduced T cell proliferation or reduced cytokine production), sustained expression of inhibitory receptors (e.g., PD-1), and a transcriptional state distinct from that of functional effector or memory T cells.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.

A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and/or changed (e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human. Native sequence human IgG1 Fc regions include a native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof. For example, a native human IgG1 Fc region amino acid sequence is provided below:

(SEQ ID NO: 36, K322 emphasized) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKC K VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK. An exemplary native human IgG4 Fc region sequence is provided below:

(SEQ ID NO: 38, S228 and L235 emphasized) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCP S CPAPEF L GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSPGK.

A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide. The variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith. For example, a variant with one amino acid K change to A at 322 position in the human IgG1 Fc amino acid sequence, IgG1-K322A Fc region, is provided below:

(SEQ ID NO: 37, K322A substitution emphasized) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKC A VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK. An exemplary variant with one amino acid S change to P at 228 position in the human IgG4 Fc amino acid sequence, IgG4P Fc region, is provided below:

(SEQ ID NO: 39, S228P substitution emphasized) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCP P CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSPGK. An exemplary variant with two amino acid changes at 228 and 235 positions in the human IgG4 Fc amino acid sequence, IgG4PE Fc region, is provided below:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCP P CPAPEF E GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40, S228P and L235E substitutions emphasized).

The term “variant” when used in relation to PD-1 or to an anti-PD-1 antibody may refer to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified sequence. For example, a PD-1 variant may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native PD-1. Also by way of example, a variant of an anti-PD-1 antibody may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native or previously unmodified anti-PD-1 antibody. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Polypeptide variants may be prepared from the corresponding nucleic acid molecules encoding the variants. In specific embodiments, the PD-1 variant or anti-PD-1 antibody variant at least retains PD-1 or anti-PD-1 antibody functional activity, respectively. In specific embodiments, an anti-PD-1 antibody variant binds PD-1 and/or is antagonistic to PD-1 activity. In specific embodiments, an anti-PD-1 antibody variant binds PD-1 and/or is agonistic to PD-1 activity. In certain embodiments, the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes PD-1 or anti-PD-1 antibody VH or VL regions or subregions, such as one or more CDRs.

The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding an anti-PD-1 antibody as described herein, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell's chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., an anti-PD-1 antibody as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted immunoglobulin bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is known (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9:457-92). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay (see, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337) can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model (see, e.g., Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-56). Antibodies with little or no ADCC activity may be selected for use.

“Antibody-dependent cellular phagocytosis” or “ADCP” refers to the destruction of target cells via monocyte or macrophage-mediated phagocytosis when immunoglobulin bound onto Fc receptors (FcRs) present on certain phagocytotic cells (e.g., neutrophils, monocytes, and macrophages) enable these phagocytotic cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell. To assess ADCP activity of a molecule of interest, an in vitro ADCP assay (see, e.g., Bracher et al., 2007, J. Immunol. Methods 323:160-71) can be performed. Useful phagocytotic cells for such assays include peripheral blood mononuclear cells (PBMC), purified monocytes from PBMC, or U937 cells differentiated to the mononuclear type. Alternatively or additionally, ADCP activity of the molecule of interest may be assessed in vivo, for example, in an animal model (see, e.g., Wallace et al., 2001, J. Immunol. Methods 248:167-82). Antibodies with little or no ADCP activity may be selected for use.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native sequence human FcR. Moreover, an exemplary FcR is one that binds an IgG antibody (e.g., a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof (see, e.g., Daeron, 1997, Annu. Rev. Immunol. 15:203-34). Various FcRs are known (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9:457-92; Capel et al., 1994, Immunomethods 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med. 126:330-41). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (see, e.g., Guyer et al., 1976, J. Immunol. 117:587-93; and Kim et al., 1994, Eu. J. Immunol. 24:2429-34). Antibody variants with improved or diminished binding to FcRs have been described (see, e.g., WO 2000/42072; U.S. Pat. Nos. 7,183,387; 7,332,581; and 7,335,742; Shields et al. 2001, J. Biol. Chem. 9(2):6591-604).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay (see, e.g., Gazzano-Santoro et al., 1996, J. Immunol. Methods 202:163) may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased Clq binding capability have been described (see, e.g., U.S. Pat. No. 6,194,551; WO 1999/51642; Idusogie et al., 2000, J. Immunol. 164: 4178-84). Antibodies with little or no CDC activity may be selected for use.

A PD-1 polypeptide “extracellular domain” or “ECD” refers to a form of the PD-1 polypeptide that is essentially free of the transmembrane and cytoplasmic domains. For example, a PD-1 polypeptide ECD may have less than 1% of such transmembrane and/or cytoplasmic domains and can have less than 0.5% of such domains.

The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

A “modification” of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/position. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (e.g., generally fewer than 5, 4, or 3) amino acids adjacent to said residue/position, and/or deletion of said residue/position.

An “epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds, such as a localized region on the surface of an antigen, such as a PD-1 polypeptide, a PD-1 polypeptide fragment, that is capable of being bound to one or more antigen binding regions of an antibody, and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human), that is capable of eliciting an immune response. An epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a polypeptide to which an antibody binds as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure. Induced epitopes are formed when the three dimensional structure of the protein is in an altered conformation, such as following activation or binding of another protein or ligand. In certain embodiments, a PD-1 epitope is a three-dimensional surface feature of a PD-1 polypeptide. In other embodiments, a PD-1 epitope is linear feature of a PD-1 polypeptide. Generally an antigen has several or many different epitopes and may react with many different antibodies.

An antibody binds “an epitope,” “essentially the same epitope,” or “the same epitope” as a reference antibody, when the two antibodies recognize identical, overlapping, or adjacent epitopes in a three-dimensional space. The most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping, or adjacent epitopes in a three-dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent, or enzyme labels.

“Epitope mapping” is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. “Epitope binning” is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, using competition assays combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct binding specificities.

A “PD-1-mediated disease,” “PD-1-mediated disorder,” and “PD-1-mediated condition” are used interchangeably and refer to any disease, disorder, or condition that is completely or partially caused by or is the result of PD-1 signaling and/or alternatively any disease, disorder, or condition in which it is desirable to mimic or augment PD-1 signaling (e.g., in vitro or in vivo effects).

An “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with a disease, disorder, or condition, including, for example, rheumatoid arthritis, psoriasis, Crohn's disease, multiple sclerosis, atopic dermatitis, lupus, or Sjögren's syndrome. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., an antibody provided herein or any other agent described herein) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder, or condition, and/or a symptom related thereto (e.g., rheumatoid arthritis, psoriasis, Crohn's disease, multiple sclerosis, atopic dermatitis, lupus, or Sjögren's syndrome). A “therapeutically effective amount” of a substance/molecule/agent of the present disclosure (e.g., an anti-PD-1 antibody) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule/agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term “therapeutically effective amount” refers to an amount of an antibody or other agent (e.g., drug) effective to “treat” a disease, disorder, or condition, in a subject or mammal.

A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing, delaying, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., rheumatoid arthritis, psoriasis, Crohn's disease, multiple sclerosis, lupus, or Sjögren's syndrome). Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, disorder, or condition, a prophylactically effective amount may be less than a therapeutically effective amount. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

“Chronic” administration refers to administration of the agent(s) in a continuous mode (e.g., for a period of time such as days, weeks, months, or years) as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term “carrier” can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral compositions, including formulations, can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990). Compositions, including pharmaceutical compounds, may contain an anti-PD-1 antibody, for example, in isolated or purified form, together with a suitable amount of carriers.

The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacoveia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

“Polyclonal antibodies” as used herein refer to an antibody population generated in an immunogenic response to a protein having many epitopes and thus includes a variety of different antibodies directed to the same or different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002)).

An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

“Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. “Oligonucleotide,” as used herein, refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. A cell that produces an anti-PD-1 antibody of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Suitable host cells are disclosed below.

Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5′ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3′ to the 3′ end of the RNA transcript are referred to as “downstream sequences.”

The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., rheumatoid arthritis, psoriasis, Crohn's disease, multiple sclerosis, lupus, or Sjögren's syndrome).

The term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset, or spread of a PD-1-mediated disease and/or symptom related thereto in a subject. In certain embodiments, the term “prophylactic agent” refers to an anti-PD-1 antibody as described herein.

As used herein, a “prophylactically effective serum titer” is the serum titer of a PD-1 antibody, e.g., a PD-1 antibody as described herein, in a subject (e.g., a human), that totally or partially inhibits the development, recurrence, onset, or spread of a PD-1-mediated disease, disorder, or condition, and/or symptom related thereto in the subject.

In certain embodiments, a “therapeutically effective serum titer” is the serum titer of a PD-1 antibody, e.g., a PD-1 antibody as described herein, in a subject (e.g., a human), that reduces the severity, the duration, and/or the symptoms associated with a PD-1-mediated disease, disorder, or condition, in the subject.

The term “recombinant antibody” refers to an antibody that is prepared, expressed, created, or isolated by recombinant means. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor et al., 1992, Nucl. Acids Res. 20:6287-95), or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies can have variable and constant regions, including those derived from human germline immunoglobulin sequences (See Kabat et al., supra). In certain embodiments, however, such recombinant antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

The term “serum titer” refers to an average serum titer in a subject from multiple samples (e.g., at multiple time points) or in a population of at least 10, at least 20, at least 40 subjects, up to about 100, 1000, or more.

The term “side effects” encompasses unwanted and/or adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., a prophylactic or therapeutic agent) might be harmful, uncomfortable, or risky. Examples of side effects include, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, loss of appetite, rashes or swellings at the site of administration, flu-like symptoms such as fever, chills, and fatigue, digestive tract problems, and allergic reactions. Additional undesired effects experienced by patients are numerous and known in the art. Many are described in Physician's Desk Reference (68th ed. 2014).

The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having a PD-1-mediated disease, disorder, or condition. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing a PD-1-mediated disease, disorder, or condition.

“Substantially all” refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

The term “therapeutic agent” refers to any agent that can be used in treating, preventing, or alleviating a disease, disorder, or condition, including in the treatment, prevention, or alleviation of one or more symptoms of a PD-1-mediated disease, disorder, or condition and/or a symptom related thereto. In certain embodiments, a therapeutic agent refers to an anti-PD-1 antibody as described herein.

The term “therapy” refers to any protocol, method, and/or agent that can be used in the prevention, management, treatment, and/or amelioration of a PD-1-mediated disease, disorder, or condition. In certain embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment, and/or amelioration of a PD-1-mediated disease, disorder, or condition, known to one of skill in the art such as medical personnel.

The term “detectable probe” refers to a composition that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, that provide a detectable signal via its activity.

The term “detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an anti-PD-1 antibody as described herein, in a sample or subject. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and/or measured (e.g., by quantitation).

The term “diagnostic agent” refers to a substance administered to a subject that aids in the diagnosis of a disease, disorder, or condition. Such substances can be used to reveal, pinpoint, and/or define the localization of a disease causing process. In certain embodiments, a diagnostic agent includes a substance that is conjugated to an anti-PD-1 antibody as described herein, that when administered to a subject or contacted with a sample from a subject aids in the diagnosis of a PD-1-mediated disease.

The term “encoding nucleic acid” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.

The term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See, also, Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990), which is hereby incorporated by reference in its entirety.

In the context of a peptide or polypeptide, the term “fragment” as used herein refers to a peptide or polypeptide that comprises less than the full length amino acid sequence. Such a fragment may arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of a residue(s) from the amino acid sequence. Fragments may, for example, result from alternative RNA splicing or from in vivo protease activity. In certain embodiments, PD-1 fragments or anti-PD-1 antibody fragments include polypeptides comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least contiguous 100 amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, or at least 950 contiguous amino acid residues of the amino acid sequence of a PD-1 polypeptide or an anti-PD-1 antibody. In a specific embodiment, a fragment of a PD-1 polypeptide or an anti-PD-1 antibody retains at least 1, at least 2, at least 3, or more functions of the polypeptide or antibody.

The terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of the disease. In certain embodiments, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an antibody provided herein) to “manage” a PD-1-mediated disease, one or more symptoms thereof, so as to prevent the progression or worsening of the disease.

The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

“Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an anti-PD-1 antibody as described herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art. When a disease, disorder, condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptoms thereof. When a disease, disorder, condition, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder, condition, or symptoms thereof.

In the context of a polypeptide, the term “analog” as used herein refers to a polypeptide that possesses a similar or identical function as a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody but does not necessarily comprise a similar or identical amino acid sequence of a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody, or possess a similar or identical structure of a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the followings: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody provided herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody (or VH or VL region thereof) described herein at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2001); and Maniatis et al., Molecular Cloning: A Laboratory Manual (1982)); or (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoding a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody (or VH or VL region thereof) described herein. A polypeptide with similar structure to a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody provided herein refers to a polypeptide that has a similar secondary, tertiary, or quaternary structure of a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody provided herein. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

In the context of a polypeptide, the term “derivative” as used herein refers to a polypeptide that comprises an amino acid sequence of a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an antibody that binds to a PD-1 polypeptide which has been altered by the introduction of amino acid residue substitutions, deletions, or additions. The term “derivative” as used herein also refers to a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an antibody that binds to a PD-1 polypeptide which has been chemically modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody may be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, chemical cleavage, formulation, metabolic synthesis of tunicamycin, linkage to a cellular ligand or other protein, etc. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide. Further, a derivative of a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a PD-1 polypeptide, a fragment of a PD-1 polypeptide, or an anti-PD-1 antibody provided herein.

The term “composition” is intended to encompass a product containing the specified ingredients (e.g., an antibody provided herein) in, optionally, the specified amounts.

4.3 Compositions and Methods of Making the Same

Provided herein are methods of managing, preventing, or treating an immune disorder in a subject, comprising administering to a subject a therapeutically effective amount of an antibody that binds to a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope.

In certain embodiments of the methods, the antibodies provided herein bind to human and/or cyno PD-1. In one embodiment, the PD-1 antibodies bind to human PD-1. In one embodiment, the PD-1 antibodies bind to cyno PD-1. In one embodiment, the PD-1 antibodies bind to both human PD-1 and cyno PD-1. In other embodiments, the antibodies provided herein do not bind to rodent PD-1.

In some embodiments of the methods, the anti-PD-1 antibodies bind to the extracellular domain (ECD) of PD-1. In certain embodiments, the anti-PD-1 antibodies bind to an epitope in the ECD of PD-1, which is distinct from the PD-L1 binding site. In certain embodiments, the anti-PD-1 antibodies bind to an epitope in the ECD of PD-1, which is distinct from the PD-L2 biding site. In certain embodiments, the anti-PD-1 antibodies bind to an epitope in the ECD of PD-1, which is distinct from both the PD-L1 and PD-L2-binding site.

In still other embodiments of the methods, the antibodies competitively block an anti-PD-1 antibody provided herein from binding to a PD-1 polypeptide.

In another embodiment of the methods, the antibodies compete for binding to a PD-1 polypeptide with an anti-PD-1 antibody provided herein.

In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L1 to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L2 to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L1 or PD-L2 to a PD-1 polypeptide.

In some embodiments, the anti-PD-1 antibodies do not compete with PD-L1 for binding to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not compete with PD-L2 for binding to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not compete with PD-L1 or PD-L2 for binding to a PD-1 polypeptide.

In certain embodiments, binding of PD-L1 to PD-1 is not inhibited by the antibody. In other embodiments, binding of PD-L2 to PD-1 is not inhibited by the antibody. In specific embodiments, neither binding of PD-L1 to PD-1 nor binding of PD-L2 to PD-1 is inhibited by the antibody.

The anti-PD-1 antibodies provided herein can also be conjugated or recombinantly fused, e.g., to a diagnostic agent or detectable agent. Further provided are compositions comprising an anti-PD-1 antibody.

4.3.1 Anti-PD-1 Antibodies

In one embodiment, the present disclosure provides anti-PD-1 antibodies that may find use herein as therapeutic agents. In another embodiment, the present disclosure provides anti-PD-1 antibodies that may find use herein as diagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having improved affinity or other properties.

In some embodiments, provided herein are antibodies that bind to PD-1, including a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope. In certain embodiments, the antibodies provided herein bind to human and/or cyno PD-1. In other embodiments, the antibodies provided herein do not bind to rodent PD-1 (e.g., a mouse PD-1). In one embodiment, an antibody provided herein binds to human PD-1. In another embodiment, an antibody provided herein binds to cyno PD-1. In another embodiment, an antibody provided herein binds to human PD-1 and cyno PD-1. In some embodiments, an antibody provided herein binds to human PD-1 and does not bind to a rodent PD-1 (e.g., a mouse PD-1). In some embodiments, an antibody provided herein binds to cyno PD-1 and does not bind to a rodent PD-1 (e.g., a mouse PD-1). In some embodiments, an antibody provided herein binds to human PD-1, binds to a cyno PD-1, and does not bind to a rodent PD-1 (e.g., a mouse PD-1). In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L1 to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L2 to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L1 or PD-L2 to a PD-1 polypeptide. In other embodiments, the anti-PD-1 antibodies are humanized antibodies (e.g., comprising human constant regions) that bind PD-1, including a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope.

In certain embodiments, the anti-PD-1 antibody comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the murine monoclonal antibodies provided herein. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 is as defined by the IMGT numbering system. In other embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 is as defined by the Kabat numbering system. In certain embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 is as defined by the AbM numbering system. In other embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 is as defined by the Chothia system. In yet other embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 is as defined by the Contact numbering system. Accordingly, in some embodiments, the isolated antibody or functional fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from: (a) the antibody PD1AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Tables 1-2.

TABLE 1 VL CDR Amino Acid Sequences VL CDR1 VL CDR2 VL CDR3 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) PD1AB-1 KSGQSVLYSSN WASTRES HQYLYSWT QKNFLA (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 1) PD1AB-2 KSSQSVLYSSN WASTRES HQYLYSWT NKNYLA (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO:7) PD1AB-3 KSGQSVLYSSNQ WASTRES HQYLYSWT KNFLA (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 1) PD1AB-4 KSSQSVLYSSNN WASTRES HQYLYSWT KNYLA (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 7) PD1AB-5 KSSQSVLYSSNN WASTRES HQYLYSWT KNYLA SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 7) PD1AB-6 KSGQSVLYSSNQ WASTRES HQYLYSWT KNFLA (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 1)

TABLE 2 VH CDR Amino Acid Sequences VH CDR1 (SEQ VH CDR2 VH CDR3 Antibody ID NO:) (SEQ ID NO:) (SEQ ID NO:) PD1AB-1 GFNIKDTYMH RIDPANGDRK SGPVYYYGSSYVMDY (SEQ ID (SEQ ID NO: 5) (SEQ ID NO: 6) NO: 4) PD1AB-2 GFNIKDTYMH RIDPANGDRK SGPVYYYGSSYVMDY (SEQ ID (SEQ ID NO: 5) (SEQ ID NO: 6) NO: 4) PD1AB-3 GFNIKDTYMH RIDPANGDRK SGPVYYYGSSYVMDY (SEQ ID (SEQ ID NO: 5) (SEQ ID NO: 6) NO: 4) PD1AB-4 GFNIKDTYMH RIDPANGDRK SGPVYYYGSSYVMDY (SEQ ID (SEQ ID NO: 5) (SEQ ID NO: 6) NO: 4) PD1AB-5 GFNIKDTYMH RIDPANGDRK SGPVYYYGSSYVMDY (SEQ ID (SEQ ID NO: 5) (SEQ ID NO: 6) NO: 4) PD1AB-6 GFNIKDTYMH RIDPANGDRK SGPVYYYGSSYVMDY (SEQ ID (SEQ ID NO: 5) (SEQ ID NO: 6) NO: 4)

In some embodiments, an antibody provided herein comprises or consists of six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-2. In some embodiments, an antibody provided herein can comprise fewer than six CDRs. In some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-2. In some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of the monoclonal antibody selected from the group consisting of: (a) the antibody PD1AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, and (f) the antibody PD1AB-6, described herein. Accordingly, in some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs of anyone of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-2.

In some embodiments, the antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 2. In other embodiments, the antibodies provided herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 1. In yet other embodiments, the antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 2 and one or more VL CDRs listed in Table 1. Accordingly, in some embodiments, the antibodies comprise a VH CDR1 having an amino acid sequence of SEQ ID NO:4. In some embodiments, the antibodies comprise a VH CDR2 having an amino acid sequence of SEQ ID NO:5. In some embodiments, the antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO:6. In some embodiments, the antibodies comprise a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from any one of the VH CDR1, VH CDR2, VH CDR3 amino acid sequence(s) as depicted in Table 2. In some embodiments, the antibodies comprise a VL CDR1 having an amino acid sequence of any one of SEQ ID NOS: 1 and 7. In another embodiment, the antibodies comprise a VL CDR2 having an amino acid sequence of SEQ ID NO:2. In some embodiments, the antibodies comprise a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In some embodiments, the antibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from any one of the VL CDR1, VL CDR2, VL CDR3 amino acid sequences as depicted in Table 1.

In certain embodiments, the antibodies provided herein comprise a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO:1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3.

In certain embodiments, the antibodies provided herein comprise a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6; and a VL region comprising: (1) a VL CDR1 having an amino acid of SEQ ID NOS:7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3.

In some embodiments, the antibodies provided herein comprise a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6.

In other embodiments, the antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO:1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3.

In some embodiments, the antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NOS: 7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3.

Also described herein are antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-2. In particular, provided herein is an antibody comprising a VH CDR1 (SEQ ID NO:4) and a VL CDR1 (SEQ ID NOS: 1 or 7). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4) and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4) and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5) and a VL CDR1 (SEQ ID NOS:1 or 7). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5) and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5) and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR3 (SEQ ID NO:6) and a VL CDR1 (SEQ ID NOS:1 or 7). In other embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6) and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6) and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), and a VL CDR1 (SEQ ID NOS:1 or 7). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), and a VL CDR3 (SEQ ID NOS:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR1 (SEQ ID NOS:1 or 7). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), and a VL CDR1 (SEQ ID NOS: 1 or 7). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR1 (SEQ ID NOS: 1 or 7), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS: 1 or 7), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In some embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR1 (SEQ ID NOS: 1 or 7). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 1-2.

In yet another aspect, the CDRs disclosed herein include consensus sequences derived from groups of related antibodies (see, e.g., Tables 1-2). As described herein, a “consensus sequence” refers to amino acid sequences having conserved amino acids common among a number of sequences and variable amino acids that vary within a given amino acid sequences.

In some embodiments, the isolated antibody or functional fragment thereof provided herein further comprises one, two, three, and/or four heavy chain FRs and/or one, two, three, and/or four light chain FRs from: (a) the antibody PD1AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Tables 3-4.

TABLE 3 VL FR Amino Acid Sequences VL FR1 VL FR2 VL FR3 VL FR4 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) PD1AB-1 DIVMTQSPDSLAVS WYQQKPGQPPKLLIY GVPDRFSGSGSGTDFT FGQGTKLEIKR LGERATINC (SEQ ID NO: 15) LTISSLQAEDVAVYYC (SEQ ID NO: 17) (SEQ ID NO: 14) (SEQ ID NO: 16) PD1AB-2 DIVMTQSPDSLAVS WYQQKPGQPPKLLIY GVPDRFSGSGSGTDFT FGQGTKLEIKR LGERATINC (SEQ ID NO: 15) LTISSLQAEDVAVYYC (SEQ ID NO: 17) (SEQ ID NO: 14) (SEQ ID NO: 16) PD1AB-3 DIVMTQSPDSLAVS WYQQKPGQPPKLLIY GVPDRFSGSGSGTDFT FGQGTKLEIKR LGERATINC (SEQ ID NO: 15) LTISNLQAEDVAVYYC (SEQ ID NO: 17) (SEQ ID NO: 14) (SEQ ID NO: 18) PD1AB-4 DIVMTQSPDSLAVS WYQQKPGQPPKLLIY GVPDRFSGSGSGTDFT FGQGTKLEIKR LGERATINC (SEQ ID NO: 15) LTISSLQAEDVAVYYC (SEQ ID NO: 17) (SEQ ID NO: 14) (SEQ ID NO: 16) PD1AB-5 DIVMTQSPDSLAVS WYQQKPGQPPKLLIY GVPDRFSGSGSGTDFT FGQGTKLEIKR LGERATINC (SEQ ID NO: 15) LTISSLQAEDVAVYYC (SEQ ID NO: 17) (SEQ ID NO: 14) (SEQ ID NO: 16) PD1AB-6 DIVMTQSPDSLAVS WYQQKPGQPPKLLIY GVPDRFSGSGSGTDFT FGQGTKLEIKR LGERATINC (SEQ ID NO: 15) LTISSLQAEDVAVYYC (SEQ ID NO: 17) (SEQ ID NO: 14) (SEQ ID NO: 16)

TABLE 4 VH FR Amino Acid Sequences VH FR1 VH FR2 VH FR3 VH FR4 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) PD1AB-1 EVQLVQSGAEVKKP WVQQAPGKGLEWMG YDPKFQGRVTITADTS WGQGTTVTVSS GATVKISCKVS (SEQ ID NO: 20) TDTAYMELSSLRSEDT (SEQ ID NO: 22) (SEQ ID NO: 19) AVYYCAR (SEQ ID NO: 21) PD1AB-2 EVQLVQSGAEVKKP WVQQAPGKGLEWMG YDPKFQGRVTITADTS WGQGTTVTVSS GATVKISCKVS (SEQ ID NO: 20) TDTAYMELSSLRSEDT (SEQ ID NO: 22) (SEQ ID NO: 19) AVYYCAR (SEQ ID NO: 21) PD1AB-3 EVQLVQSGAEVKKP WVQQAPGKGLEWMG YDPKFQGRVTITADTS WGQGTTVTVSS GATVKISCKVS (SEQ ID NO: 20) TNTAYMELSSLRSEDT (SEQ ID NO: 22) (SEQ ID NO: 19) AVYYCAR (SEQ ID NO: 23) PD1AB-4 EVQLVQSGAEVKKP WVQQAPGKGLEWMG YDPKFQGRVTITADTS WGQGTTVTVSS GATVKISCKVS (SEQ ID NO: 20) TNTAYMELSSLRSEDT (SEQ ID NO: 22) (SEQ ID NO: 19) AVYYCAR (SEQ ID NO: 23) PD1AB-5 EVQLVQSGAEVKKP WVQQAPGKGLEWMG YDPKFQGRVTITADTS WGQGTTVTVSS GATVKISCKAS (SEQ ID NO: 20) TDTAYMELSSLRSEDT (SEQ ID NO: 22) (SEQ ID NO: 24) AVYYCAR (SEQ ID NO: 21) PD1AB-6 EVQLVQSGAEVKKP WVQQAPGKGLEWMG YDPKFQGRVTITADTS WGQGTTVTVSS GATVKISCKAS (SEQ ID NO: 20) TDTAYMELSSLRSEDT (SEQ ID NO: 22) (SEQ ID NO: 24) AVYYCAR (SEQ ID NO: 21)

In certain embodiments, the isolated antibody or functional fragment thereof provided herein further comprises one, two, three, and/or four heavy chain FRs from: (a) the antibody PD AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Table 4. In some embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-1. In some embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-2. In other embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-3. In certain embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-4. In other embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-5. In another embodiment, the antibody heavy chain FR(s) is from the antibody PD1AB-6.

In some embodiments, the isolated antibody or functional fragment thereof provided herein further comprises one, two, three, and/or four light chain FRs from: (a) the antibody PD AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Table 3. In some embodiments, the antibody light chain FR(s) is from the antibody PD1AB-1. In some embodiments, the antibody light chain FR(s) is from the antibody PD1AB-2. In other embodiments, the antibody light chain FR(s) is from the antibody PD1AB-3. In certain embodiments, the antibody light chain FR(s) is from the antibody PD1AB-4. In other embodiments, the antibody light chain FR(s) is from the antibody PD1AB-5. In another embodiment, the antibody light chain FR(s) is from the antibody PD1AB-6.

In certain embodiments, an antibody of fragment thereof described herein comprises a VH region that comprises: (1) a VH FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 19 and 24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:21 and 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, an antibody of fragment thereof described herein comprises a VH region that comprises: (1) a VH FR1 having an amino acid of SEQ ID NO: 19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, an antibody of fragment thereof described herein comprises a VH region that comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO: 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, an antibody of fragment thereof described herein comprises a VH region that comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO: 24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, an antibody of fragment thereof described herein comprises a VH region that comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO: 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In specific embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4.

Accordingly, in some embodiments, the humanized antibody comprises a VH region that includes a VH FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 19 and 24. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR1 having an amino acid sequence of SEQ ID NO: 19. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR1 having an amino acid sequence of SEQ ID NO:24. In some embodiments, the humanized antibody comprises a VH region that includes a VH FR2 having an amino acid sequence of SEQ ID NO: 20. In some embodiments, the humanized antibody comprises a VH region that includes a VH FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:21 and 23. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR3 having an amino acid sequence of SEQ ID NO:21. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR3 having an amino acid sequence of SEQ ID NO:23. In other embodiments, the humanized antibody comprises a VH region that includes a VH FR4 having an amino acid sequence of SEQ ID NO:22.

In certain embodiments, an antibody of fragment thereof described herein comprises a VL region that comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO:15; (3) a VL FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:16 and 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NOS: 16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In other embodiments, the VL region that comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO:15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17.

Accordingly, in some embodiments, the humanized antibody comprises a VL region that includes a VL FR1 having an amino acid sequence of SEQ ID NO: 14. In certain embodiments, the humanized antibody comprises a VL region that includes a VL FR2 having an amino acid sequence of SEQ ID NO: 15. In other embodiments, the humanized antibody comprises a VL region that includes a VL FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:16 and 18. In one embodiment, the humanized antibody comprises a VL region that includes a VL FR3 having an amino acid sequence of SEQ ID NOS: 16. In other embodiments, the humanized antibody comprises a VL region that includes a VL FR3 having an amino acid sequence of SEQ ID NO: 18. In yet other embodiments, the humanized antibody comprises a VL region that includes a VL FR4 having an amino acid sequence of SEQ ID NO:17.

In certain embodiments, an antibody of fragment thereof described herein comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 19 and 24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:21 and 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO:15; (3) a VL FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:16 and 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3 and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3 and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, an antibody of fragment thereof comprises a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

Also described herein are antibodies comprising one or more (e.g., one, two, three, or four) VH FRs and one or more (e.g., one, two, three, or four) VL FRs listed in Tables 3-4. In particular, provided herein is an antibody comprising a VH FR1 (SEQ ID NOS: 19 or 24) and a VL FR1 (SEQ ID NO: 14). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24) and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24) and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR1 (SEQ ID NO: 14). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR2 (SEQ ID NO: 15). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR4 (SEQ ID NO: 17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR1 (SEQ ID NO:14). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR2 (SEQ ID NO:15). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR1 (SEQ ID NO: 14). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR2 (SEQ ID NO: 15). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR1 (SEQ ID NO: 14). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR4 (SEQ ID NO: 17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR1 (SEQ ID NO: 14). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR2 (SEQ ID NO:15). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR3 (SEQ ID NOS: 16 or 18). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO:15). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR2 (SEQ ID NO:15) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NO:19 or 24), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO: 15) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15) and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15) and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO: 17). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR1 (SEQ ID NO:14). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR2 (SEQ ID NO: 15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO: 14). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO: 15). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO: 14). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO: 14). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO: 15). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS: 16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO: 14). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO: 15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO: 15), a VL FR3 (SEQ ID NOS: 16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises any combination thereof of the VH FRs (SEQ ID NOS: 19-24) and the VL FRs (SEQ ID NOS:14-18) listed in Tables 3-4.

In some embodiments, the antibodies provided herein comprise a VH region or VH domain. In other embodiments, the antibodies provided herein comprise a VL region or VL domain. In certain embodiments, the antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region. In yet other embodiments, the antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region selected from the group consisting of SEQ ID NOS: 8-13 as set forth in Tables 5-6. In still other embodiments, the antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6, as set forth in Tables 5-6.

In certain embodiments, the antibodies provided herein comprise a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6; and a VL region selected from the group consisting of SEQ ID NOS:8-10 as set forth in Table 5. In some embodiments, the VL region has an amino acid sequence of SEQ ID NO:8. In other embodiments, the VL region has an amino acid sequence of SEQ ID NO:9. In some embodiments, the VL region has an amino acid sequence of SEQ ID NO:10.

In other embodiments, the antibodies provided herein comprise a VH region selected from the group consisting of SEQ ID NOS: 11-13 as set forth in Table 6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 and 7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In yet some embodiments, the antibodies provided herein comprise a VH region selected from the group consisting of SEQ ID NOS:11-13 as set forth in Table 6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In still other embodiments, the antibodies provided herein comprise a VH region selected from the group consisting of SEQ ID NOS:11-13 as set forth in Table 6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO:7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In some embodiments, the VH region has an amino acid sequence of SEQ ID NO: 11. In some embodiments, the VH region has an amino acid sequence of SEQ ID NO:12. In some embodiments, the VH region has an amino acid sequence of SEQ ID NO: 13.

TABLE 5 VL Domain Amino Acid Sequences Antibody VL (SEQ ID NO:) PD1AB-1 DIVMTQSPDSLAVSLGERATINCKSGQSVLYSSNQKNFLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCHQYLYSWTFGQGTKLEIKR (SEQ ID NO: 8) PD1AB-2 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCHQYLYSWTFGQGTKLEIKR (SEQ ID NO: 9) PD1AB-3 DIVMTQSPDSLAVSLGERATINCKSGQSVLYSSNQKNFLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISNLQAEDVAVYYCHQYLYSWTFGQGTKLEIKR (SEQ ID NO: 10) PD1AB-4 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCHQYLYSWTFGQGTKLEIKR (SEQ ID NO: 9) PD1AB-5 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCHQYLYSWTFGQGTKLEIKR (SEQ ID NO: 9) PD1AB-6 DIVMTQSPDSLAVSLGERATINCKSGQSVLYSSNQKNFLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCHQYLYSWTFGQGTKLEIKR (SEQ ID NO: 8)

TABLE 6 VH Domain Amino Acid Sequences Antibody VH (SEQ ID NO:) PD1AB-1 EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEWMGRIDPANGDRKYDPKFQGRV TITADTSTDTAYMELSSLRSEDTAVYYCARSGPVYYYGSSYVMDYWGQGTTVTVSS (SEQ ID NO: 11) PD1AB-2 EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEWMGRIDPANGDRKYDPKFQGRV TITADTSTDTAYMELSSLRSEDTAVYYCARSGPVYYYGSSYVMDYWGQGTTVTVSS (SEQ ID NO: 11) PD1AB-3 EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEWMGRIDPANGDRKYDPKFQGRV TITADTSTNTAYMELSSLRSEDTAVYYCARSGPVYYYGSSYVMDYWGQGTTVTVSS (SEQ ID NO: 12) PD1AB-4 EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEWMGRIDPANGDRKYDPKFQGRV TITADTSTNTAYMELSSLRSEDTAVYYCARSGPVYYYGSSYVMDYWGQGTTVTVSS (SEQ ID NO: 12) PD1AB-5 EVQLVQSGAEVKKPGATVKISCKASGFNIKDTYMHWVQQAPGKGLEWMGRIDPANGDRKYDPKFQGRV TITADTSTDTAYMELSSLRSEDTAVYYCARSGPVYYYGSSYVMDYWGQGTTVTVSS (SEQ ID NO: 13) PD1AB-6 EVQLVQSGAEVKKPGATVKISCKASGFNIKDTYMHWVQQAPGKGLEWMGRIDPANGDRKYDPKFQGRV TITADTSTDTAYMELSSLRSEDTAVYYCARSGPVYYYGSSYVMDYWGQGTTVTVSS (SEQ ID NO: 13)

Also provided herein are isolated nucleic acid molecules encoding an immunoglobulin heavy chain, light chain, VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of anti-PD-1 antibodies that bind to a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope. The exemplary nucleic acid sequences for the VL region and the VH region of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, and PD1AB-6 are shown in Tables 7-8.

TABLE 7 VL Nucleic Acid Sequences Antibody Nucleotide sequences PD1AB-1 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAA CTGCAAGTCCGGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTTCTTGGCCTGGTACCAGC AGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCT GACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAAGCTGA AGATGTGGCAGTTTATTACTGTCATCAATACCTCTACTCGTGGACGTTTGGCCAGGGGACCAAGC TGGAGATCAAACGGAC (SEQ ID NO: 25) PD1AB-2 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAA CTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGC AGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCT GACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAAGCTGA AGATGTGGCAGTTTATTACTGTCATCAATACCTCTACTCGTGGACGTTTGGCCAGGGGACCAAGC TGGAGATCAAACGGAC (SEQ ID NO: 26) PD1AB-3 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAA CTGCAAGTCCGGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTTCTTGGCCTGGTACCAGC AGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCT GACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAACCTGCAAGCTGA AGATGTGGCAGTTTATTACTGTCATCAATACCTCTACTCGTGGACGTTTGGCCAGGGGACCAAGC TGGAGATCAAACGGAC (SEQ ID NO: 27) PD1AB-4 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAA CTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGC AGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCT GACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAAGCTGA AGATGTGGCAGTTTATTACTGTCATCAATACCTCTACTCGTGGACGTTTGGCCAGGGGACCAAGC TGGAGATCAAACGGAC (SEQ ID NO: 26) PD1AB-5 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAA CTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGC AGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCT GACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAAGCTGA AGATGTGGCAGTTTATTACTGTCATCAATACCTCTACTCGTGGACGTTTGGCCAGGGGACCAAGC TGGAGATCAAACGGAC (SEQ ID NO: 26) PD1AB-6 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAA CTGCAAGTCCGGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTTCTTGGCCTGGTACCAGC AGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCT GACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAAGCTGA AGATGTGGCAGTTTATTACTGTCATCAATACCTCTACTCGTGGACGTTTGGCCAGGGGACCAAGC TGGAGATCAAACGGAC (SEQ ID NO: 25)

TABLE 8 VH Nucleic Acid Sequences Antibody Nucleotide sequences PD1AB-1 GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTG CAAGGTTTCTGGATTCAACATTAAAGACACGTATATGCACTGGGTGCAACAGGCCCCTGGAAAAG GGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTGATAGGAAATATGACCCGAAGTTCCAG GGCAGAGTCACCATAACCGCGGACACGTCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACGGCCGTGTATTACTGTGCTAGATCAGGCCCTGTTTATTACTACGGTAGTAGCT ACGTTATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA (SEQ ID NO: 28) PD1AB-2 GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTG CAAGGTTTCTGGATTCAACATTAAAGACACGTATATGCACTGGGTGCAACAGGCCCCTGGAAAAG GGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTGATAGGAAATATGACCCGAAGTTCCAG GGCAGAGTCACCATAACCGCGGACACGTCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACGGCCGTGTATTACTGTGCTAGATCAGGCCCTGTTTATTACTACGGTAGTAGCT ACGTTATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA (SEQ ID NO: 28) PD1AB-3 GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTG CAAGGTTTCTGGATTCAACATTAAAGACACGTATATGCACTGGGTGCAACAGGCCCCTGGAAAAG GGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTGATAGGAAATATGACCCGAAGTTCCAG GGCAGAGTCACCATAACCGCGGACACGTCTACAAACACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACGGCCGTGTATTACTGTGCTAGATCAGGCCCTGTTTATTACTACGGTAGTAGCT ACGTTATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA (SEQ ID NO: 29) PD1AB-4 GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTG CAAGGTTTCTGGATTCAACATTAAAGACACGTATATGCACTGGGTGCAACAGGCCCCTGGAAAAG GGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTGATAGGAAATATGACCCGAAGTTCCAG GGCAGAGTCACCATAACCGCGGACACGTCTACAAACACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACGGCCGTGTATTACTGTGCTAGATCAGGCCCTGTTTATTACTACGGTAGTAGCT ACGTTATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA (SEQ ID NO: 29) PD1AB-5 GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTG CAAGGCTTCTGGATTCAACATTAAAGACACGTATATGCACTGGGTGCAACAGGCCCCTGGAAAAG GGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTGATAGGAAATATGACCCGAAGTTCCAG GGCAGAGTCACCATAACCGCGGACACGTCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACGGCCGTGTATTACTGTGCTAGATCAGGCCCTGTTTATTACTACGGTAGTAGCT ACGTTATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA (SEQ ID NO: 30) PD1AB-6 GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTG CAAGGCTTCTGGATTCAACATTAAAGACACGTATATGCACTGGGTGCAACAGGCCCCTGGAAAAG GGCTTGAGTGGATGGGAAGGATTGATCCTGCGAATGGTGATAGGAAATATGACCCGAAGTTCCAG GGCAGAGTCACCATAACCGCGGACACGTCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACGGCCGTGTATTACTGTGCTAGATCAGGCCCTGTTTATTACTACGGTAGTAGCT ACGTTATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA (SEQ ID NO: 30)

In some embodiments, an antibody provided herein has a VH and a VL amino acid sequence of PD1AB-1. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 11, and a VL amino acid sequence of SEQ ID NO:8.

In other embodiments, an antibody provided herein has a VH and a VL amino acid sequence of PD1AB-2. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 11, and a VL amino acid sequence of SEQ ID NO:9.

In some embodiments, an antibody provided herein has a VH and a VL amino acid sequence of PD1AB-3. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 12, and a VL amino acid sequence of SEQ ID NO: 10.

In other embodiments, an antibody provided herein has a VH and a VL amino acid sequence of PD1AB-4. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 12, and a VL amino acid sequence of SEQ ID NO:9.

In some embodiments, an antibody provided herein has a VH and a VL amino acid sequence of PD1AB-5. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 13, and a VL amino acid sequence of SEQ ID NO:9.

In other embodiments, an antibody provided herein has a VH and a VL amino acid sequence of PD1AB-6. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 13, and a VL amino acid sequence of SEQ ID NO:8.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the light chain comprises a constant region having an amino acid sequence of:

(SEQ ID NO: 41) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In other embodiments, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG1 Fc region having an amino acid sequence of:

(SEQ ID NO: 36, K322 emphasized) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKC K VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK. In some embodiments, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain does not comprise a human IgG Fc region having an amino acid sequence of SEQ ID NO:36.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG1-K322A Fc region having an amino acid sequence of:

(SEQ ID NO: 37, K322A substitution emphasized) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKC A VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG4 Fc region having an amino acid sequence of:

(SEQ ID NO: 38, S228 and L235 emphasized) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCP S CPAPEF L GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG4P Fc region having an amino acid sequence of:

(SEQ ID NO: 39, S228P substitution emphasized) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCP P CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSPGK.

In yet another embodiment, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG4PE Fc region having an amino acid sequence of:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCP P CPAPEF E GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40, S228P and L235E substitutions emphasized). In some embodiments, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain does not comprise a human IgG4PE Fc region having an amino acid sequence of SEQ ID NO:40.

In still another embodiment, an antibody or antigen-binding fragment thereof described herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the light chain comprises a constant region having an amino acid sequence of SEQ ID NO:41; and the heavy chain comprises an Fc region having an amino acid sequence selected from the group consisting of SEQ ID NOS:36-40.

In certain embodiments, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the light chain comprises an amino acid sequence as follows:

(SEQ ID NO: 31, LC_PD1AB-6-IgG1) DIVMTQSPDSLAVSLGERATINCKSGQSVLYSSNQKNFLAWYQQKPGQPP KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLYS WTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

In some embodiments, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence as follows:

(SEQ ID NO: 32, HC_PD1AB-6-IgG1, K322 emphasized) EVQLVQSGAEVKKPGATVKISCKASGFNIKDTYMHWVQQAPGKGLEWMGR IDPANGDRKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARSG PVYYYGSSYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKC K VSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK.

In other embodiments, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence as follows:

EVQLVQSGAEVKKPGATVKISCKASGFNIKDTYMHWVQQAPGKGLEWMGR IDPANGDRKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARSG PVYYYGSSYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKC A VSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 33, HC_PD1AB-6-IgG1-K322A, K322A substitution emphasized).

In another embodiment, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence as follows:

EVQLVQSGAEVKKPGATVKISCKASGFNIKDTYMHWVQQAPGKGLEWMGR IDPANGDRKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARSG PVYYYGSSYVMDYWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCP   CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS PGK  (SEQ ID NO: 34, HC_PD1AB-6-IgG4P, IgG4P Fc backbone italicized and underlined).

In yet another embodiment, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence as follows:

EVQLVQSGAEVKKPGATVKISCKASGFNIKDTYMHWVQQAPGKGLEWMGR IDPANGDRKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARSG PVYYYGSSYVMDYWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCP   CPAPEF   GGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSPGK . (SEQ ID NO: 35, HC_PD1AB-6-IgG4PE, IgG4PE Fc backbone italicized and underlined)

In one particular embodiment, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:32.

In another particular embodiment, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:33.

In yet another particular embodiment, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:34.

In still another particular embodiment, an antibody provided herein, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:35.

In yet another aspect, antibodies are provided that compete with one of the exemplified antibodies or functional fragments for binding to PD-1. Such antibodies may also bind to the same epitope as one of the herein exemplified antibodies, or an overlapping epitope. Antibodies and fragments that compete with or bind to the same epitope as the exemplified antibodies are expected to show similar functional properties. The exemplified antigen-binding proteins and fragments include those with the VH and VL regions, and CDRs provided herein, including those in Tables 1-6. Thus, as a specific example, the antibodies that are provided include those that compete with an antibody comprising: (a) 1, 2, 3, 4, 5, or all 6 of the CDRs listed for an antibody listed in Tables 1-2; (b) a VH and a VL selected from the VH and the VL regions listed for an antibody listed in Tables 5-6; or (c) two light chains and two heavy chains comprising a VH and a VL as specified for an antibody listed in Tables 5-6. In some embodiments, the antibody is PD1AB-1. In some embodiments, the antibody is PD1AB-2. In some embodiments, the antibody is PD1AB-3. In some embodiments, the antibody is PD1AB-4. In some embodiments, the antibody is PD1AB-5. In some embodiments, the antibody is PD1AB-6.

In another aspect, antibodies or antigen-binding fragments thereof provided herein bind to a region, including an epitope, of human PD-1 or cyno PD-1. For example, in some embodiments, an antibody provided herein binds to a region of human PD-1 (SEQ ID NO:42) comprising amino acid residues 33 to 109 of human PD-1. In still another aspect, antibodies provided herein bind to a specific epitope of human PD-1.

In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one of residues 100-109 (SEQ ID NO:43) within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one of residues 100-105 (SEQ ID NO:44) within an amino acid sequence of SEQ ID NO:42.

In particular embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one residue selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one residue selected from the group consisting of L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to two or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In other embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to three or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to four or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In one embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to five or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to six or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In yet another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to seven or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In still another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to eight or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to nine or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In other embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to all ten residues from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42.

In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to N33 within an amino acid sequence of SEQ ID NO:42. In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to T51 within an amino acid sequence of SEQ ID NO:42. In a particular embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to S57 within an amino acid sequence of SEQ ID NO:42. In one specific embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to L100 within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to N102 within an amino acid sequence of SEQ ID NO:42. In other embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to G103 within an amino acid sequence of SEQ ID NO:42. In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to R104 within an amino acid sequence of SEQ ID NO:42. In yet another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to G103 and R104 within an amino acid sequence of SEQ ID NO:42. In still another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to D105 within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to H107 within an amino acid sequence of SEQ ID NO:42. In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to 5109 within an amino acid sequence of SEQ ID NO:42. Any combination of two, three, four, five, six, seven, eight, nine, ten or more of the above-referenced amino acid PD-1 binding sites is also contemplated.

In one aspect, described herein are antibodies that specifically bind to PD-1 and can modulate PD-1 activity and/or expression (e.g., activate PD-1 signaling and/or inhibit PD-1 expression). In certain embodiments, a PD-1 agonist is provided herein that is an antibody provided herein that specifically binds to an ECD of human PD-1, and activates (e.g., partially activates) at least one PD-1 activity (e.g., inhibiting cytokine production). In certain embodiments, a PD-1 agonist provided herein is an antibody provided herein that specifically binds to an ECD of human PD-1, and downregulates PD-1 expression. In certain embodiments, described herein are antibodies that specifically bind to PD-1 and that (a) attenuate T cell activity, e.g., as determined by inhibition of cytokine production; and/or (b) downregulate PD-1 expression in a cell. In certain embodiments, described herein are antibodies that specifically bind to PD-1 and that (a) attenuate T cell activity, e.g., as determined by inhibition of cytokine production; (b) downregulate PD-1 expression in a cell; and/or (c) do not inhibit PD-L1 and/or PD-L2 binding to PD-1. In certain embodiments, the antibodies that specifically bind to PD-1 bind to an ECD of human PD-1, or an epitope of an ECD of human PD-1 thereof. In certain embodiments, the antibodies specifically bind to an epitope of an ECD of human PD-1 that is distinct from the PD-L1 binding site. In certain embodiments, the antibodies specifically bind to an epitope of an ECD of human PD-1 that is distinct from the PD-L2 binding site. In certain embodiments, the antibodies specifically bind to an epitope of an ECD of human PD-1 that is distinct from both the PD-L1 and PD-L2 binding sites. In certain embodiments, binding of PD-L1 to PD-1 is not inhibited by the antibody. In other embodiments, binding of PD-L2 to PD-1 is not inhibited by the antibody. In specific embodiments, neither binding of PD-L1 to PD-1 nor binding of PD-L2 to PD-1 is inhibited by the antibody.

PD-1 activity can relate to any activity of PD-1 such as those known or described in the art. PD-1 activity and PD-1 signaling are used interchangeably herein. In certain aspects, PD-1 activity is induced by PD-1 ligand (e.g., PD-L) binding to PD-1. Expression levels of PD-1 can be assessed by methods described herein or known to one of skill in the art (e.g., Western blotting, ELISA, immunohistochemistry, or flow cytometry). In certain embodiments, described herein are antibodies that specifically bind to PD-1 and decrease PD-1 expression. In certain embodiments, described herein are antibodies that specifically bind to PD-1 and attenuate T cell activity. In certain embodiments, described herein are antibodies that specifically bind to PD-1 and inhibit cytokine production. In certain embodiments, described herein are antibodies that specifically bind to PD-1 and activate (e.g., partially activate) PD-1 signaling. In certain embodiments, the antibodies that specifically bind to PD-1 bind to an ECD of human PD-1, or an epitope of an ECD of human PD-1 thereof. In certain embodiments, the antibodies specifically bind to an epitope of an ECD of human PD-1 that is distinct from the PD-L1 binding site. In certain embodiments, the antibodies specifically bind to an epitope of an ECD of human PD-1 that is distinct from the PD-L2 binding site. In certain embodiments, the antibodies specifically bind to an epitope of an ECD of human PD-1 that is distinct from both the PD-L1 and PD-L2 binding sites. In certain embodiments, binding of PD-L1 to PD-1 is not inhibited by the antibody. In other embodiments, binding of PD-L2 to PD-1 is not inhibited by the antibody. In specific embodiments, neither binding of PD-L1 to PD-1 nor binding of PD-L2 to PD-1 is inhibited by the antibody.

In certain embodiments, an anti-PD-1 antibody provided herein attenuates (e.g., partially attenuate) T cell activity. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 10%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 15%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 20%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 25%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 30%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 35%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 40%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 45%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 50%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 55%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 60%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 65%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 70%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 75%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 80%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 85%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 90%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 95%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 98%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 99%. In some embodiments, an anti-PD-1 antibody provided herein attenuates T cell activity by at least about 100%. In certain embodiments, an anti-PD-1 antibody provided herein can attenuate (e.g., partially attenuate) T cell activity by at least about 25% to about 65%. In specific embodiments, the T cell activity attenuation is assessed by methods described herein. In some embodiments, the T cell activity attenuation is assessed by methods known to one of skill in the art. In certain embodiments, the T cell activity attenuation is relative to T cell activity in the presence of stimulation without any anti-PD-1 antibody. In certain embodiments, the T cell activity attenuation is relative to T cell activity in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In one embodiment, attenuation of T cell activity is indicated by inhibition of T cell proliferation. In specific embodiments, the T cell proliferation is CD4+ T cell proliferation. In certain embodiments, the T cell proliferation is CD8+ T cell proliferation. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 10%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 15%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 20%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 25%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 30%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 35%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 40%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 45%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 50%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 55%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 60%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 65%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 70%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 75%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 80%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 85%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 90%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 95%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 98%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 99%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell proliferation by at least about 100%. In certain embodiments, an anti-PD-1 antibody provided herein can inhibit (e.g., partially inhibit) T cell proliferation by at least about 25% to about 65%. In specific embodiments, the T cell proliferation is assessed by methods described herein. In some embodiments, the T cell proliferation is assessed by methods known to one of skill in the art. In certain embodiments, the T cell proliferation is relative to T cell proliferation in the presence of stimulation without any anti-PD-1 antibody. In certain embodiments, the T cell proliferation is relative to T cell proliferation in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD AB-3, PD AB-4, PD AB-5, or PD AB-6) specifically bind to PD-1 and inhibit CD4+ T cell proliferation. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD4+ T cell proliferation by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibits CD4+ T cell proliferation by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of CD4+ T cell proliferation is assessed by methods described herein. In other embodiments, the inhibition of CD4+ T cell proliferation is assessed by methods known to one of skill in the art (e.g., thymidine incorporation assay, MTT assay, or cell proliferation biomarker assay). In a specific embodiment, the inhibition of CD4+ T cell proliferation is assessed by thymidine incorporation assay. In another embodiment, the inhibition of CD4+ T cell proliferation is assessed by MTT assay. In yet another embodiment, the inhibition of CD4+ T cell proliferation is assessed by cell proliferation biomarker assay. In a specific embodiment, CD4+ T cell proliferation is inhibited relative to CD4+ T cell proliferation in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the CD4+ T cell proliferation is inhibited relative to CD4+ T cell proliferation in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits CD4+ T cell proliferation. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD4+ T cell proliferation with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., thymidine incorporation assay, MTT assay, or cell proliferation biomarker assay). In a specific embodiment, the EC₅₀ is assessed by thymidine incorporation assay. In another embodiment, the EC₅₀ is assessed by MTT assay. In yet another embodiment, the EC₅₀ is assessed by cell proliferation biomarker assay.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit CD8+ T cell proliferation. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits CD8+ T cell proliferation by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibits CD8+ T cell proliferation by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of CD8+ T cell proliferation is assessed by methods described herein. In other embodiments, the inhibition of CD8+ T cell proliferation is assessed by methods known to one of skill in the art (e.g., thymidine incorporation assay, MTT assay, or cell proliferation biomarker assay). In a specific embodiment, the inhibition of CD8+ T cell proliferation is assessed by thymidine incorporation assay. In another embodiment, the inhibition of CD8+ T cell proliferation is assessed by MTT assay. In yet another embodiment, the inhibition of CD8+ T cell proliferation is assessed by cell proliferation biomarker assay. In a specific embodiment, CD8+ T cell proliferation is inhibited relative to CD8+ T cell proliferation in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the CD8+ T cell proliferation is inhibited relative to CD8+ T cell proliferation in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits CD8+ T cell proliferation. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits CD8+ T cell proliferation with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., thymidine incorporation assay, MTT assay, or cell proliferation biomarker assay). In a specific embodiment, the EC₅₀ is assessed by thymidine incorporation assay. In another embodiment, the EC₅₀ is assessed by MTT assay. In yet another embodiment, the EC₅₀ is assessed by cell proliferation biomarker assay.

In one embodiment, attenuation of T cell activity is indicated by upregulation of expression of an inhibitory receptor on the surface of T cells that is not the PD-1 receptor (e.g., LAG-3, CTLA-4, or TIM-3). In some embodiments, the inhibitory receptor is selected from the group consisting of LAG-3, CTLA-4, or TIM-3, or any combination thereof. In one embodiment, the inhibitory receptor is LAG-3. In another embodiment, the inhibitory receptor is CTLA-4. In yet another embodiment, the inhibitory receptor is TIM-3. In certain embodiments, the inhibitory receptor is LAG-3 and CTLA-4. In some embodiments, the inhibitory receptor is LAG-3 and TIM-3. In yet other embodiments, the inhibitory receptor is CTLA-4 and TIM-3. In still other embodiments, the inhibitory receptor is LAG-3, CTLA-4, or TIM-3.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and upregulate the expression of the LAG-3 receptor. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates LAG-3 expression by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and upregulate LAG-3 expression by at least about 25% or 35%, optionally to about 75%. In some embodiments, the upregulation of LAG-3 expression is assessed by methods described herein. In other embodiments, the upregulation of LAG-3 expression is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the upregulation of LAG-3 expression is assessed by flow cytometry. In another embodiment, the upregulation of LAG-3 expression is assessed by Western blotting. In yet another embodiment, the upregulation of LAG-3 expression is assessed by Northern blotting. In still another embodiment, the upregulation of LAG-3 expression is assessed by RT-PCR. In a specific embodiment, LAG-3 expression is upregulated relative to LAG-3 expression in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the LAG-3 expression is upregulated relative to LAG-3 expression in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) upregulates LAG-3 expression. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates LAG-3 expression with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and upregulate the expression of the CTLA-4 receptor. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates CTLA-4 expression by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and upregulates CTLA-4 expression by at least about 25% or 35%, optionally to about 75%. In some embodiments, the upregulation of CTLA-4 expression is assessed by methods described herein. In other embodiments, the upregulation of CTLA-4 expression is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the upregulation of CTLA-4 expression is assessed by flow cytometry. In another embodiment, the upregulation of CTLA-4 expression is assessed by Western blotting. In yet another embodiment, the upregulation of CTLA-4 expression is assessed by Northern blotting. In still another embodiment, the upregulation of CTLA-4 expression is assessed by RT-PCR. In a specific embodiment, CTLA-4 expression is upregulated relative to CTLA-4 expression in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the CTLA-4 expression is upregulated relative to CTLA-4 expression in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) upregulates CTLA-4 expression. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates CTLA-4 expression with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and upregulate the expression of the TIM-3 receptor. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates TIM-3 expression by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and upregulate TIM-3 expression by at least about 25% or 35%, optionally to about 75%. In some embodiments, the upregulation of TIM-3 expression is assessed by methods described herein. In other embodiments, the upregulation of TIM-3 expression is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the upregulation of TIM-3 expression is assessed by flow cytometry. In another embodiment, the upregulation of TIM-3 expression is assessed by Western blotting. In yet another embodiment, the upregulation of TIM-3 expression is assessed by Northern blotting. In still another embodiment, the upregulation of TIM-3 expression is assessed by RT-PCR. In a specific embodiment, TIM-3 expression is upregulated relative to TIM-3 expression in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the TIM-3 expression is upregulated relative to TIM-3 expression in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) upregulates TIM-3 expression. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates TIM-3 expression with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In one embodiment, attenuation of T cell activity is indicated by inhibition of T cell recall response. In some embodiments, the T cell recall response is measured by CD4+ T cell proliferation. In other embodiments, the T cell recall response is measured by CD25 upregulation. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 10%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 15%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 20%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 25%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 30%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 35%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 40%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 45%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 50%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 55%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 60%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 65%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 70%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 75%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 80%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 85%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 90%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 95%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 98%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 99%. In some embodiments, an anti-PD-1 antibody provided herein inhibits T cell recall response by at least about 100%. In certain embodiments, an anti-PD-1 antibody provided herein can inhibit (e.g., partially inhibit) T cell recall response by at least about 25% to about 65%. In specific embodiments, the T cell recall response is assessed by methods described herein. In some embodiments, the T cell recall response is assessed by methods known to one of skill in the art (e.g., CD4+ T cell proliferation assay or CD25 upregulation assay). In certain embodiments, the T cell recall response is relative to T cell recall response in the presence of stimulation without any anti-PD-1 antibody. In certain embodiments, the T cell recall response is relative to T cell recall response in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In some embodiments, attenuation of T cell activity is indicated by downregulation of T cell activation biomarkers. In one embodiment, the T cell activation biomarker is CD25. In another embodiment, the T cell activation biomarker is CD69.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and downregulate the expression of CD25. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD25 expression by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and downregulate CD25 expression by at least about 25% or 35%, optionally to about 75%. In some embodiments, the downregulation of CD25 expression is assessed by methods described herein. In other embodiments, the downregulation of CD25 expression is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the downregulation of CD25 expression is assessed by flow cytometry. In another embodiment, the downregulation of CD25 expression is assessed by Western blotting. In yet another embodiment, the downregulation of CD25 expression is assessed by Northern blotting. In still another embodiment, the downregulation of CD25 expression is assessed by RT-PCR. In a specific embodiment, CD25 expression is downregulated relative to CD25 expression in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the CD25 expression is downregulated relative to CD25 expression in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) downregulates CD25 expression. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD25 expression with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and downregulate the expression of CD69. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates CD69 expression by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and downregulate CD69 expression by at least about 25% or 35%, optionally to about 75%. In some embodiments, the downregulation of CD69 expression is assessed by methods described herein. In other embodiments, the downregulation of CD69 expression is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the downregulation of CD69 expression is assessed by flow cytometry. In another embodiment, the downregulation of CD69 expression is assessed by Western blotting. In yet another embodiment, the downregulation of CD69 expression is assessed by Northern blotting. In still another embodiment, the downregulation of CD69 expression is assessed by RT-PCR. In a specific embodiment, CD69 expression is downregulated relative to CD69 expression in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the CD69 expression is downregulated relative to CD69 expression in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) downregulates CD69 expression. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates CD69 expression with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In some embodiments, attenuation of T cell activity is indicated by upregulation of regulatory T cell biomarkers. In one embodiment, the regulatory T cell biomarker is Foxp3.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and upregulate the expression of Foxp3. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and upregulates Foxp3 expression by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and upregulate Foxp3 expression by at least about 25% or 35%, optionally to about 75%. In some embodiments, the upregulation of Foxp3 expression is assessed by methods described herein. In other embodiments, the upregulation of Foxp3 expression is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the upregulation of Foxp3 expression is assessed by flow cytometry. In another embodiment, the upregulation of Foxp3 expression is assessed by Western blotting. In yet another embodiment, the upregulation of Foxp3 expression is assessed by Northern blotting. In still another embodiment, the upregulation of Foxp3 expression is assessed by RT-PCR. In a specific embodiment, Foxp3 expression is upregulated relative to Foxp3 expression in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, the Foxp3 expression is upregulated relative to FOXP3 expression in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) upregulates Foxp3 expression. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein upregulates Foxp3 expression with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In one embodiment, attenuation of T cell activity is indicated by increase of T cell exhaustion. In certain embodiments, the increase of T cell exhaustion is measured by reduction of IL-7R^(hi) T cells. In some embodiments, the increase of T cell exhaustion is measured by increase of IL-7R^(lo) T cells. In other embodiments, the increase of T cell exhaustion is measured by increase of LAG-3 expression. In yet other embodiments, the increase of T cell exhaustion is measured by increase of CTLA-4 expression. In still other embodiments, the increase of T cell exhaustion is measured by increase of TIM-3 expression. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 10%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 15%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 20%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 25%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 30%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 35%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 40%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 45%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 50%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 55%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 60%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 65%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 70%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 75%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 80%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 85%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 90%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 95%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 98%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 99%. In some embodiments, an anti-PD-1 antibody provided herein increases T cell exhaustion by at least about 100%. In certain embodiments, an anti-PD-1 antibody provided herein can inhibit (e.g., partially inhibit) T cell recall response by at least about 25% to about 65%. In specific embodiments, the T cell exhaustion is assessed by methods described herein. In some embodiments, the T cell exhaustion is assessed by methods known to one of skill in the art (e.g., IL-7R^(hi) T cell flow cytometry; IL-7R^(lo) T cell flow cytometry; LAG-3 expression flow cytometry, western blot, northern blot, RT-PCR; CTLA-4 expression flow cytometry, western blot, northern blot, RT-PCR; or TIM-3 expression flow cytometry, western blot, northern blot, RT-PCR). In certain embodiments, the T cell exhaustion is relative to T cell exhaustion in the presence of stimulation without any anti-PD-1 antibody. In certain embodiments, the T cell exhaustion is relative to T cell exhaustion in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1). In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In yet other embodiments, the T cells are CD4+ T cells and CD8+ T cells.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and reduces IL-7R^(hi) T cells. In one embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and reduces IL-7R^(hi) T cells by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and reduce IL-7R^(hi) T cells by at least about 25% or 35%, optionally to about 75%. In some embodiments, the reduction of IL-7R^(hi) T cells is assessed by methods described herein. In other embodiments, the reduction of IL-7R^(hi) T cells is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the reduction of IL-7R^(hi) T cells is assessed by flow cytometry. In another embodiment, the reduction of IL-7R^(hi) T cells is assessed by Western blotting. In yet another embodiment, the reduction of IL-7R^(hi) T cells is assessed by Northern blotting. In still another embodiment, the reduction of IL-7R^(hi) T cells is assessed by RT-PCR. In a specific embodiment, IL-7R^(hi) T cells are reduced relative to IL-7R^(hi) T cells in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, IL-7R^(hi) T cells are reduced relative to IL-7R^(hi) T cells in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1). In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In yet other embodiments, the T cells are CD4+ T cells and CD8+ T cells.

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) reduces IL-7R^(hi) T cells. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R′ T cells with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein reduces IL-7R^(hi) T cells with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR. In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In yet other embodiments, the T cells are CD4+ T cells and CD8+ T cells.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and increase IL-7R^(lo) T cells. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases IL-7R^(lo) T cells by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and increase IL-7R^(lo) T cells by at least about 25% or 35%, optionally to about 75%. In some embodiments, the increase of IL-7R^(lo) T cells is assessed by methods described herein. In other embodiments, the increase of IL-7R^(lo) T cells is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the increase of IL-7R^(lo) T cells is assessed by flow cytometry. In another embodiment, the increase of IL-7R^(lo) T cells is assessed by Western blotting. In yet another embodiment, the increase of IL-7R^(lo) T cells is assessed by Northern blotting. In still another embodiment, the increase of IL-7R^(lo) T cells is assessed by RT-PCR. In a specific embodiment, IL-7R^(lo) T cells are increased relative to IL-7R^(lo) T cells in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, IL-7R^(lo) T cells are increased relative to IL-7R^(lo) T cells in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1). In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In yet other embodiments, the T cells are CD4+ T cells and CD8+ T cells.

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) increases IL-7R^(lo) T cells. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo)T cells with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein increases IL-7R^(lo) T cells with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR. In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In yet other embodiments, the T cells are CD4+ T cells and CD8+ T cells.

In one embodiment, attenuation of T cell activity is indicated by increase of regulatory T cells. In some embodiments, attenuation of T cell activity is indicated by increase of Foxp3+ regulatory T cells. In some embodiments, attenuation of T cell activity is indicated by increase of CD25+Foxp3+ regulatory T cells.

In certain embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and increase CD25+Foxp3+ regulatory T cells. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and increases CD25+Foxp3+ regulatory T cells by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and increase CD25+Foxp3+ regulatory T cells by at least about 25% or 35%, optionally to about 75%. In some embodiments, the increase of CD25+Foxp3+ regulatory T cells is assessed by methods described herein. In other embodiments, the increase of CD25+Foxp3+ regulatory T cells is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the increase of CD25+Foxp3+ regulatory T cells is assessed by flow cytometry. In another embodiment, the increase of CD25+Foxp3+ regulatory T cells is assessed by Western blotting. In yet another embodiment, the increase of CD25+Foxp3+ regulatory T cells is assessed by Northern blotting. In still another embodiment, the increase of CD25+Foxp3+ regulatory T cells is assessed by RT-PCR. In a specific embodiment, CD25+Foxp3+ regulatory T cells are increased relative to CD25+Foxp3+ regulatory T cells in the presence of stimulation without any anti-PD-1 antibody. In other embodiments, CD25+Foxp3+ regulatory T cells are increased relative to CD25+Foxp3+ regulatory T cells in the presence of stimulation with an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) increases CD25+Foxp3+ regulatory T cells. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein increases CD25+Foxp3+ regulatory T cells with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In one embodiment, attenuation of T cell activity is indicated by inhibition of a cytokine secretion. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-17, IFN-γ, or any combination thereof. In certain embodiments, the cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, IFN-γ, and TNF-α. In certain embodiments, the cytokine is IL-1. In some embodiments, the cytokine is IL-2. In other embodiments, the cytokine is IL-6. In another embodiment, the cytokine is IL-12. In other embodiments, the cytokine is IL-17. In yet other embodiments, the cytokine is IL-22. In still other embodiments, the cytokine is IL-23. In some embodiments, the cytokine is GM-CSF. In other embodiments, the cytokine is IFN-γ. In yet other embodiments, the cytokine is TNF-α. In certain embodiments, the cytokine is IL-2 and IL-17. In some embodiments, the cytokine is IL-2 and IFN-γ. In yet other embodiments, the cytokine is IL-17 and IFN-γ. In still other embodiments, the cytokine is IL-2, IL-17, and IFN-γ.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit IL-2 secretion (e.g., from a cell, for example, T cells). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-2 secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IL-2 secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IL-2 secretion is assessed by methods described herein. In other embodiments, the inhibition of IL-2 secretion is assessed by methods known to one of skill in the art (e.g., MesoScale™ Discovery (MSD) multiplex assay). In a specific embodiment, the IL-2 secretion is inhibited relative to IL-2 secretion in the absence of anti-PD-1 antibody. In other embodiments, the IL-2 secretion is inhibited relative to IL-2 secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits IL-2 secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-2 secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit IL-17 secretion (e.g., from a cell, for example, T cells). In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 5%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 10%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 15%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 20%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 25%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 35%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 40%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 50%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 55%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 65%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 70%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 80%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 85%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 95%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 98%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-17 secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IL-17 secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IL-17 secretion is assessed by methods described herein. In other embodiments, the inhibition of IL-17 secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the IL-17 secretion is inhibited relative to IL-17 secretion in the absence of anti-PD-1 antibody. In other embodiments, the IL-17 secretion is inhibited relative to IL-17 secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits IL-17 secretion. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 50 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 40 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 20 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 1 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 0.75 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 0.5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 0.1 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 0.05 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 0.005 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at most about 0.001 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 50 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 0.5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 0.005 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-17 secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit IFN-γ secretion (e.g., from a cell, for example, T cells). In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 10%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 15%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 25%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 30%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 40%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 45%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 50%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 55%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 60%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 65%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 70%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 75%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 85%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 90%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 95%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 98%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IFN-γ secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IFN-γ secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IFN-γ secretion is assessed by methods described herein. In other embodiments, the inhibition of IFN-γ secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the IFN-γ secretion is inhibited relative to IFN-γ secretion in the absence of anti-PD-1 antibody. In other embodiments, the IFN-γ secretion is inhibited relative to IFN-γ secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits IFN-γ secretion. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 50 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 40 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 20 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 10 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 0.75 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 0.05 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 0.005 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at most about 0.001 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 50 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 40 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 20 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 10 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 1 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 0.75 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 0.5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 0.1 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 0.05 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 0.005 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IFN-γ secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit IL-1 secretion (e.g., from a cell, for example, T cells). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-1 secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IL-1 secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IL-1 secretion is assessed by methods described herein. In other embodiments, the inhibition of IL-1 secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the IL-1 secretion is inhibited relative to IL-1 secretion in the absence of anti-PD-1 antibody. In other embodiments, the IL-1 secretion is inhibited relative to IL-1 secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits IL-1 secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-1 secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit IL-6 secretion (e.g., from a cell, for example, T cells). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-6 secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IL-6 secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IL-6 secretion is assessed by methods described herein. In other embodiments, the inhibition of IL-6 secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the IL-6 secretion is inhibited relative to IL-6 secretion in the absence of anti-PD-1 antibody. In other embodiments, the IL-6 secretion is inhibited relative to IL-6 secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits IL-6 secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-6 secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD AB-3, PD AB-4, PD AB-5, or PD AB-6) specifically bind to PD-1 and inhibit IL-12 secretion (e.g., from a cell, for example, T cells). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-12 secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IL-12 secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IL-12 secretion is assessed by methods described herein. In other embodiments, the inhibition of IL-12 secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the IL-12 secretion is inhibited relative to IL-12 secretion in the absence of anti-PD-1 antibody. In other embodiments, the IL-12 secretion is inhibited relative to IL-12 secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD AB-5, or PD1AB-6) inhibits IL-12 secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-12 secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit IL-22 secretion (e.g., from a cell, for example, a T cell). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-22 secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IL-22 secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IL-22 secretion is assessed by methods described herein. In other embodiments, the inhibition of IL-22 secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the IL-22 secretion is inhibited relative to IL-22 secretion in the absence of anti-PD-1 antibody. In other embodiments, the IL-22 secretion is inhibited relative to IL-22 secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits IL-22 secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-22 secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit IL-23 secretion (e.g., from a cell, for example, a T cell). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits IL-23 secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit IL-23 secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of IL-23 secretion is assessed by methods described herein. In other embodiments, the inhibition of IL-23 secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the IL-23 secretion is inhibited relative to IL-23 secretion in the absence of anti-PD-1 antibody. In other embodiments, the IL-23 secretion is inhibited relative to IL-23 secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits IL-23 secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits IL-23 secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit GM-CSF secretion (e.g., from a cell, for example, T cells). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits GM-CSF secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit GM-CSF secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of GM-CSF secretion is assessed by methods described herein. In other embodiments, the inhibition of GM-CSF secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the GM-CSF secretion is inhibited relative to GM-CSF secretion in the absence of anti-PD-1 antibody. In other embodiments, the GM-CSF secretion is inhibited relative to GM-CSF secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits GM-CSF secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits GM-CSF secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and inhibit TNF-α secretion (e.g., from a cell, for example, a T cell). In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 5%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 15%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 20%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 30%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 35%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 45%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 50%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 60%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 75%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 80%. In other embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 90%. In one embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 95%. In some embodiments, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and inhibits TNF-α secretion by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and inhibit TNF-α secretion by at least about 25% or 35%, optionally to about 75%. In some embodiments, the inhibition of TNF-α secretion is assessed by methods described herein. In other embodiments, the inhibition of TNF-α secretion is assessed by methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the TNF-α secretion is inhibited relative to TNF-α secretion in the absence of anti-PD-1 antibody. In other embodiments, the TNF-α secretion is inhibited relative to TNF-α secretion in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) inhibits TNF-α secretion. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein inhibits TNF-α secretion with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., MSD multiplex assay). In a specific embodiment, the EC₅₀ is assessed by MSD multiplex assay.

In specific embodiments, antibodies provided herein (e.g., any one of antibodies PD AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) specifically bind to PD-1 and downregulate PD-1 expression (e.g., in a cell, for example, T cells). In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 5%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 10%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 15%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 20%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 25%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 30%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 35%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 40%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 45%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 50%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 55%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 60%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 65%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 70%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 75%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 80%. In some embodiments, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 85%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 90%. In other embodiments, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 95%. In one embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 98%. In another embodiment, an antibody provided herein specifically binds to PD-1 and downregulates PD-1 expression by at least about 99%. In specific embodiments, antibodies provided herein specifically bind to PD-1 and downregulates PD-1 expression by at least about 25% or 35%, optionally to about 75%. In some embodiments, the downregulation of PD-1 expression is assessed by methods described herein. In other embodiments, the downregulation of PD-1 expression is assessed by methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the downregulation of PD-1 expression is assessed by flow cytometry. In another embodiment, the downregulation of PD-1 expression is assessed by Western blotting. In yet another embodiment, the downregulation of PD-1 expression is assessed by Northern blotting. In still another embodiment, the downregulation of PD-1 expression is assessed by RT-PCR. In a specific embodiment, the PD-1 expression is downregulated relative to PD-1 expression downregulation in the absence of anti-PD-1 antibody. In other embodiments, the PD-1 expression is downregulated relative to PD-1 expression downregulation in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to PD-1).

In certain embodiments, an anti-PD-1 antibody provided herein (e.g., any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6) downregulates PD-1 expression. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 40 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 30 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 10 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 5 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 0.75 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 0.5 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 0.05 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 0.01 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at most about 0.001 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 50 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 40 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 30 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 20 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 10 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 5 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 1 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 0.75 nM. In other embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 0.5 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 0.1 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 0.05 nM. In some embodiments, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 0.01 nM. In another embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 0.005 nM. In one embodiment, an anti-PD-1 antibody provided herein downregulates PD-1 expression with an EC₅₀ of at least about 0.001 nM. In specific embodiments, the EC₅₀ is assessed by methods described herein. In other embodiments, the EC₅₀ is assessed by other methods known to one of skill in the art (e.g., flow cytometry, Western blotting, Northern blotting, or RT-PCR). In a specific embodiment, the EC₅₀ is assessed by flow cytometry. In another embodiment, the EC₅₀ is assessed by Western blotting. In yet another embodiment, the EC₅₀ is assessed by Northern blotting. In still another embodiment, the EC₅₀ is assessed by RT-PCR.

In certain embodiments of the methods, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof. In another embodiment, the downregulation occurs as early as 6 hours after the contact. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact. In still another embodiment, the downregulation occurs as early as 10 hours after the contact. In one embodiment, the downregulation occurs as early as 12 hours after the contact. In another embodiment, the downregulation occurs as early as 14 hours after the contact. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact. In still another embodiment, the downregulation occurs as early as 18 hours after the contact. In one embodiment, the downregulation occurs as early as 20 hours after the contact. In another embodiment, the downregulation occurs as early as 22 hours after the contact. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact. In some embodiments, the contact is with the antibody. In other embodiments, the contact is with an antigen-binding fragment thereof.

In some embodiments, the downregulation of PD-1 expression on the surface of T cells precedes cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 6 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In still another embodiment, the downregulation occurs as early as 10 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 12 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 14 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In still another embodiment, the downregulation occurs as early as 18 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In one embodiment, the downregulation occurs as early as 20 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In another embodiment, the downregulation occurs as early as 22 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact with the antibody or antigen-binding fragment thereof, and precedes cytokine inhibition.

In other embodiments, the downregulation of PD-1 expression on the surface of T cells is concurrent with cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 6 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In still another embodiment, the downregulation occurs as early as 10 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 12 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 14 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In still another embodiment, the downregulation occurs as early as 18 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In one embodiment, the downregulation occurs as early as 20 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In another embodiment, the downregulation occurs as early as 22 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact with the antibody or antigen-binding fragment thereof, and is concurrent with cytokine inhibition.

In yet other embodiments, the downregulation of PD-1 expression on the surface of T cells is after cytokine inhibition. In one embodiment, the downregulation of PD-1 expression on the surface of T cells occurs as early as 4 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 6 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 8 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In still another embodiment, the downregulation occurs as early as 10 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 12 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 14 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 16 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In still another embodiment, the downregulation occurs as early as 18 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In one embodiment, the downregulation occurs as early as 20 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In another embodiment, the downregulation occurs as early as 22 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition. In yet another embodiment, the downregulation occurs as early as 24 hours after the contact with the antibody or antigen-binding fragment thereof, and is after cytokine inhibition.

4.3.1.1 Polyclonal Antibodies

The antibodies of the present disclosure may comprise polyclonal antibodies.

Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a PD-1 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized or to immunize the mammal with the protein and one or more adjuvants. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Ribi, CpG, Poly 1C, Freund's complete adjuvant, and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. The mammal can then be bled, and the serum assayed for PD-1 antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus. Additionally or alternatively, lymphocytes may be obtained from the immunized animal for fusion and preparation of monoclonal antibodies from hybridoma as described below.

4.3.1.2 Monoclonal Antibodies

The antibodies of the present disclosure may alternatively be monoclonal antibodies. Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., 1975, Nature 256:495-97, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Exemplary myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, Va.), and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, Calif.). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, 1984, Immunol. 133:3001-05; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as RIA or ELISA. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., 1980, Anal. Biochem. 107:220-39.

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal, for example, by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., 1993, Curr. Opinion in Immunol. 5:256-62 and Plückthun, 1992, Immunol. Revs. 130:151-88.

In some embodiments, an antibody that binds a PD-1 epitope comprises an amino acid sequence of a VH domain and/or an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes to (1) the complement of a nucleotide sequence encoding any one of the VH and/or VL domain described herein under stringent conditions (e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions which are known to those of skill in the art. See, e.g., Current Protocols in Molecular Biology Vol. I, 6.3.1-6.3.6 and 2.10.3 (Ausubel et al. eds., 1989).

In some embodiments, an antibody that binds a PD-1 epitope comprises an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding any one of the VH CDRs and/or VL CDRs depicted in Tables 1-2 under stringent conditions (e.g., hybridization to filter-bound DNA in 6×SSC at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions which are known to those of skill in the art (see, e.g., Ausubel et al., supra).

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols (O'Brien and Aitken eds., 2002). In principle, synthetic antibody clones are selected by screening phage libraries containing phages that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption/elution.

Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

Repertoires of VH and VL genes can be separately cloned by PCR and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., supra. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., 1993, EMBO J 12:725-34. Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by Hoogenboom and Winter, 1992, J. Mol. Biol. 227:381-88.

Screening of the libraries can be accomplished by various techniques known in the art. For example, PD-1 (e.g., a PD-1 polypeptide, fragment, or epitope) can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., 1990, Proteins 8:309-14 and WO 92/09690, and by use of a low coating density of antigen as described in Marks et al., 1992, Biotechnol. 10:779-83.

Anti-PD-1 antibodies can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-PD-1 antibody clone using VH and/or VL sequences (e.g., the Fv sequences), or various CDR sequences from VH and VL sequences, from the phage clone of interest and suitable constant region (e.g., Fc) sequences described in Kabat et al., supra.

In another embodiment, anti-PD-1 antibody is generated by using methods as described in Bowers et al., 2011, Proc Natl Acad Sci USA. 108:20455-60, e.g., the SHM-XHL™ platform (AnaptysBio, San Diego, Calif.). Briefly, in this approach, a fully human library of IgGs is constructed in a mammalian cell line (e.g., HEK293) as a starting library. Mammalian cells displaying immunoglobulin that binds to a target peptide or epitope are selected (e.g., by FACS sorting), then activation-induced cytidine deaminase (AID)-triggered somatic hypermutation is reproduced in vitro to expand diversity of the initially selected pool of antibodies. After several rounds of affinity maturation by coupling mammalian cell surface display with in vitro somatic hypermutation, high affinity, high specificity anti-PD-1 antibodies are generated. Further methods that can be used to generate antibody libraries and/or antibody affinity maturation are disclosed, e.g., in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Publ. Nos. 2014/0170705, 2014/0094392, 2012/0028301, 2011/0183855, and 2009/0075378, each of which are incorporated herein by reference.

4.3.1.3 Antibody Fragments

The present disclosure provides antibodies and antibody fragments that bind to PD-1. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to cells, tissues, or organs. For a review of certain antibody fragments, see Hudson et al., 2003, Nature Med. 9:129-34.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al., 1992, Bio/Technology 10:163-67). According to another approach, F(ab′)₂ fragments can be isolated directly from recombinant host cell culture. Fab and F(ab′)₂ fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). Fv and scFv have intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv (See, e.g., Borrebaeck ed., supra). The antibody fragment may also be a “linear antibody,” for example, as described in the references cited above. Such linear antibodies may be monospecific or multi-specific, such as bispecific.

Smaller antibody-derived binding structures are the separate variable domains (V domains) also termed single variable domain antibodies (sdAbs). Certain types of organisms, the camelids and cartilaginous fish, possess high affinity single V-like domains mounted on an Fc equivalent domain structure as part of their immune system. (Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49). The V-like domains (called VhH in camelids and V-NAR in sharks) typically display long surface loops, which allow penetration of cavities of target antigens. They also stabilize isolated VH domains by masking hydrophobic surface patches.

These VhH and V-NAR domains have been used to engineer sdAbs. Human V domain variants have been designed using selection from phage libraries and other approaches that have resulted in stable, high binding VL- and VH-derived domains.

Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen binding site that bind to a PD-1 epitope. The immunoglobulin molecules provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.

Variants and derivatives of antibodies include antibody functional fragments that retain the ability to bind to a PD-1 epitope. Exemplary functional fragments include Fab fragments (e.g., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab′ (e.g., an antibody fragment containing a single antigen-binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab′)₂ (e.g., two Fab′ molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab′ molecules may be directed toward the same or different epitopes); a bispecific Fab (e.g., a Fab molecule having two antigen binding domains, each of which may be directed to a different epitope); a single chain comprising a variable region, also known as, scFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked Fv, or dsFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (e.g., the variable, antigen-binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific scFv (e.g., an scFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (e.g., a dimerized scFv formed when the VH domain of a first scFv assembles with the VL domain of a second scFv and the VL domain of the first scFv assembles with the VH domain of the second scFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); and a triabody (e.g., a trimerized scFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen-binding domains may be directed towards the same or different epitopes).

4.3.1.4 Humanized Antibodies

In some embodiments, antibodies provided herein can be humanized antibodies that bind PD-1, including human and/or cyno PD-1. For example, humanized antibodies of the present disclosure may comprise one or more CDRs as shown in Tables 1-2. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.

In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the “specificity determining residues,” or SDRs (Padlan et al., 1995, FASEB J. 9:133-39). In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36:25-34).

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. For example, according to the so-called “best-fit” method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308; and Chothia et al., 1987, J. Mol. Biol. 196:901-17). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, V_(L)6 subgroup I (V_(L)6I) and V_(H) subgroup III (V_(H)III). In another method, human germline genes are used as the source of the framework regions.

In an alternative paradigm based on comparison of CDRs, called superhumanization, FR homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., 2002, J. Immunol. 169:1119-25).

It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes, and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (Lazar et al., 2007, Mol. Immunol. 44:1986-98).

In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those that are based upon the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

In the FR library approach, a collection of residue variants are introduced at specific positions in the FR followed by screening of the library to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224:487-99), or from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

In FR shuffling, whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall'Acqua et al., 2005, Methods 36:43-60). The libraries may be screened for binding in a two-step process, first humanizing VL, followed by VH. Alternatively, a one-step FR shuffling process may be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

The “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non-human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96% homologous to human germline gene antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

The “human engineering” method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk,” “moderate risk,” or “high risk” residues. The classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody's folding. The particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody's variable regions with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail in Studnicka et al., 1994, Protein Engineering 7:805-14; U.S. Pat. Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619; and PCT Publication WO 93/11794.

4.3.1.5 Human Antibodies

Human anti-PD-1 antibodies can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal anti-PD-1 antibodies of the present disclosure can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, 1984, J. Immunol. 133:3001-05; Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Boerner et al., 1991, J. Immunol. 147:86-95.

It is also possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice that express human antibody repertoires have been used to generate high-affinity human sequence monoclonal antibodies against a wide variety of potential drug targets (see, e.g., Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; U.S. Pat. Nos. 6,075,181 and 6,150,584; and Lonberg et al., 2005, Nature Biotechnol. 23:1117-25).

Alternatively, the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (e.g., such B lymphocytes may be recovered from an individual or may have been immunized in vitro) (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and U.S. Pat. No. 5,750,373).

Gene shuffling can also be used to derive human antibodies from non-human, for example, rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting” or “guided selection,” either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone (e.g., the epitope guides (imprints) the choice of the human chain partner). When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see, e.g., PCT WO 93/06213; and Osbourn et al., 2005, Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin. Examples of guided selection to humanize mouse antibodies towards cell surface antigens include the folate-binding protein present on ovarian cancer cells (see, e.g., Figini et al., 1998, Cancer Res. 58:991-96) and CD147, which is highly expressed on hepatocellular carcinoma (see, e.g., Bao et al., 2005, Cancer Biol. Ther. 4:1374-80).

A potential disadvantage of the guided selection approach is that shuffling of one antibody chain while keeping the other constant could result in epitope drift. In order to maintain the epitope recognized by the non-human antibody, CDR retention can be applied (see, e.g., Klimka et al., 2000, Br. J. Cancer. 83:252-60; and Beiboer et al., 2000, J. Mol. Biol. 296:833-49). In this method, the non-human VH CDR3 is commonly retained, as this CDR may be at the center of the antigen-binding site and may be the most important region of the antibody for antigen recognition. In some instances, however, VH CDR3 and VL CDR3, as well as VH CDR2, VL CDR2, and VL CDR1 of the non-human antibody may be retained.

4.3.1.6 Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for PD-1 and the other is for any other antigen. In some embodiments, one of the binding specificities is for PD-1, and the other is for another surface antigen expressed on cells expressing PD-1. In certain embodiments, bispecific antibodies may bind to two different epitopes of PD-1. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art, such as, by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details of generating bispecific antibodies, see, for example, Bispecific Antibodies (Kontermann ed., 2011).

4.3.1.7 Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. In certain embodiments, a multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, a multivalent antibody comprises (or consists of) four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may further comprise at least two (e.g., four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

4.3.1.9 Fc Engineering

It may be desirable to modify an anti-PD-1 antibody provided herein by Fc engineering. In certain embodiments, the modification to the Fc region of the antibody results in the decrease or elimination of an effector function of the antibody. In certain embodiments, the effector function is ADCC, ADCP, and/or CDC. In some embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP and CDC. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. For example, substitutions into human IgG1 using IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 were shown to greatly reduce ADCC and CDC (see, e.g., Armour et al., 1999, Eur. J. Immunol. 29(8):2613-24; and Shields et al., 2001, J. Biol. Chem. 276(9): 6591-604). Other Fc variants are provided elsewhere herein.

To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment), for example, as described in U.S. Pat. No. 5,739,277. Term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

4.3.1.10 Alternative Binding Agents

The present disclosure encompasses non-immunoglobulin binding agents that specifically bind to the same epitope as an anti-PD-1 antibody disclosed herein. In some embodiments, a non-immunoglobulin binding agent is identified as an agent that displaces or is displaced by an anti-PD-1 antibody of the present disclosure in a competitive binding assay. These alternative binding agents may include, for example, any of the engineered protein scaffolds known in the art. Such scaffolds may comprise one or more CDRs as shown in Tables 1-2. Such scaffolds include, for example, anticalins, which are based upon the lipocalin scaffold, a protein structure characterized by a rigid beta-barrel that supports four hypervariable loops which form the ligand binding site. Novel binding specificities may be engineered by targeted random mutagenesis in the loop regions, in combination with functional display and guided selection (see, e.g., Skerra, 2008, FEBS J. 275:2677-83). Other suitable scaffolds may include, for example, adnectins, or monobodies, based on the tenth extracellular domain of human fibronectin III (see, e.g., Koide and Koide, 2007, Methods Mol. Biol. 352: 95-109); affibodies, based on the Z domain of staphylococcal protein A (see, e.g., Nygren et al., 2008, FEBS J. 275:2668-76); DARPins, based on ankyrin repeat proteins (see, e.g., Stumpp et al., 2008, Drug. Discov. Today 13:695-701); fynomers, based on the SH3 domain of the human Fyn protein kinase (see, e.g., Grabulovski et al., 2007, J. Biol. Chem. 282:3196-204); affitins, based on Sac7d from Sulfolobus acidolarius (see, e.g., Krehenbrink et al., 2008, J. Mol. Biol. 383:1058-68); affilins, based on human y-B-crystallin (see, e.g., Ebersbach et al., 2007, J. Mol. Biol. 372:172-85); avimers, based on the A domain of membrane receptor proteins (see, e.g., Silverman et al., 2005, Biotechnol. 23:1556-61); cysteine-rich knottin peptides (see, e.g., Kolmar, 2008, FEBS J. 275:2684-90); and engineered Kunitz-type inhibitors (see, e.g., Nixon and Wood, 2006, Curr. Opin. Drug. Discov. Dev. 9:261-68). For a review, see, for example, Gebauer and Skerra, 2009, Curr. Opin. Chem. Biol. 13:245-55.

4.3.2 Antibody Variants

In some embodiments, amino acid sequence modification(s) of the antibodies that bind to PD-1 or described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the anti-PD-1 antibodies provided herein, it is contemplated that anti-PD-1 antibody variants can be prepared. For example, anti-PD-1 antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art who appreciate that amino acid changes may alter post-translational processes of the anti-PD-1 antibody, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

In some embodiments, antibodies provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the antibody. The antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.

Variations may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Alternatively, conservative (e.g., within an amino acid group with similar properties and/or side chains) substitutions may be made, so as to maintain or not significantly change the properties. Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His(H).

Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites. Accordingly, in one embodiment, an antibody or fragment thereof that binds to a PD-1 epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a murine monoclonal antibody provided herein. In one embodiment, an antibody or fragment thereof that binds to a PD-1 epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence depicted in Tables 1-6. In yet another embodiment, an antibody or fragment thereof that binds to a PD-1 epitope comprises a VH CDR and/or a VL CDR amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a VH CDR amino acid sequence depicted in Table 2 and/or a VL CDR amino acid sequence depicted in Table 1. The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315-23), or other known techniques can be performed on the cloned DNA to produce the anti-PD-1 antibody variant DNA.

Any cysteine residue not involved in maintaining the proper conformation of the anti-PD-1 antibody also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-PD-1 antibody to improve its stability (e.g., where the antibody is an antibody fragment such as an Fv fragment).

In some embodiments, an anti-PD-1 antibody molecule of the present disclosure is a “de-immunized” antibody. A “de-immunized” anti-PD-1 antibody is an antibody derived from a humanized or chimeric anti-PD-1 antibody, which has one or more alterations in its amino acid sequence resulting in a reduction of immunogenicity of the antibody, compared to the respective original non-de-immunized antibody. One of the procedures for generating such antibody mutants involves the identification and removal of T cell epitopes of the antibody molecule. In a first step, the immunogenicity of the antibody molecule can be determined by several methods, for example, by in vitro determination of T cell epitopes or in silico prediction of such epitopes, as known in the art. Once the critical residues for T cell epitope function have been identified, mutations can be made to remove immunogenicity and retain antibody activity. For review, see, for example, Jones et al., 2009, Methods in Molecular Biology 525:405-23.

4.3.2.1 In Vitro Affinity Maturation

In some embodiments, antibody variants having an improved property such as affinity, stability, or expression level as compared to a parent antibody may be prepared by in vitro affinity maturation. Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Libraries of antibodies are displayed as Fab, scFv, or V domain fragments either on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association (e.g., covalently or non-covalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies. Two or three rounds of mutation and selection using display methods such as phage display usually results in antibody fragments with affinities in the low nanomolar range. Affinity matured antibodies can have nanomolar or even picomolar affinities for the target antigen.

Phage display is a widespread method for display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to the bacteriophage coat protein. Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning.” Phage bound to antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For review, see, for example, Hoogenboom, 2002, Methods. Mol. Biol. 178:1-37; and Bradbury and Marks, 2004, J. Immunol. Methods 290:29-49.

In a yeast display system (see, e.g., Boder et al., 1997, Nat. Biotech. 15:553-57; and Chao et al., 2006, Nat. Protocols 1:755-68), the antibody may be displayed as single-chain variable fusions (scFv) in which the heavy and light chains are connected by a flexible linker. The scFv is fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to Agalp. Display of a protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen the library to select for antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling of yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variations in surface expression of the antibody can be measured through immunofluorescence labeling of either the hemagglutinin or c-Myc epitope tag flanking the scFv. Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., 1999, J. Mol. Biol. 292:949-56). An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cells, taking advantage of endoplasmic reticulum chaperones and quality-control machinery. Once maturation is complete, antibody affinity can be conveniently “titrated” while displayed on the surface of the yeast, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is the potentially smaller functional library size than that of other display methods; however, a recent approach uses the yeast cells' mating system to create combinatorial diversity estimated to be 10¹⁴ in size (see, e.g., U.S. Pat. Publication 2003/0186374; and Blaise et al., 2004, Gene 342:211-18).

In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system. The DNA library coding for a particular library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to surface-bound ligand, allowing simultaneous isolation of the antibody and its encoding mRNA through affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back into cDNA, which can then undergo mutagenesis and be used in the next round of selection (see, e.g., Fukuda et al., 2006, Nucleic Acids Res. 34:e127). In mRNA display, a covalent bond between antibody and mRNA is established using puromycin as an adaptor molecule (Wilson et al., 2001, Proc. Natl. Acad. Sci. USA 98:3750-55).

As these methods are performed entirely in vitro, they provide two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, for example, by non-proofreading polymerases, as no library must be transformed after any diversification step.

In a mammalian cell display system (see, e.g., Bowers et al., 2011, Proc Natl Acad Sci USA. 108:20455-60), a fully human library of IgGs is constructed based on germline sequence V-gene segments joined to prerecombined D(J) regions. Full-length V regions for heavy chain and light chain are assembled with human heavy chain and light chain constant regions and transfected into a mammalian cell line (e.g., HEK293). The transfected library is expanded and subjected to several rounds of negative selection against streptavidin (SA)-coupled magnetic beads, followed by a round of positive selection against SA-coupled magnetic beads coated with biotinylated target protein, peptide fragment, or epitope. Positively selected cells are expanded, and then sorted by rounds of FACS to isolate single cell clones displaying antibodies that specifically bind to the target protein, peptide fragment, or epitope. Heavy and light chain pairs from these single cell clones are retransfected with AID for further maturation. Several rounds of mammalian cell display, coupled with AID-triggered somatic hypermutation, generate high specificity, high affinity antibodies.

Diversity may also be introduced into the CDRs or the whole V genes of the antibody libraries in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via a high or low level of mutagenesis or targeting isolated hot spots of somatic hypermutations (see, e.g., Ho et al., 2005, J. Biol. Chem. 280:607-17) or residues suspected of affecting affinity on experimental basis or structural reasons. In a specific embodiment, somatic hypermutation is performed by AID-triggered somatic hypermutation, e.g., using the SHM-XEL™ platform (AnaptysBio, San Diego, Calif.). Random mutations can be introduced throughout the whole V gene using E. coli mutator strains, error-prone replication with DNA polymerases (see, e.g., Hawkins et al., 1992, J. Mol. Biol. 226:889-96), or RNA replicases. Diversity may also be introduced by replacement of regions that are naturally diverse via DNA shuffling or similar techniques (see, e.g., Lu et al., 2003, J. Biol. Chem. 278:43496-507; U.S. Pat. Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into framework-region residues (see, e.g., Bond et al., 2005, J. Mol. Biol. 348:699-709) employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, e.g., U.S. Pat. Publication No. 2004/0005709). Additional methods of generating diversity in CDRs are disclosed, for example, in U.S. Pat. No. 7,985,840. Further methods that can be used to generate antibody libraries and/or antibody affinity maturation are disclosed, e.g., in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Publ. Nos. 2014/0170705, 2014/0094392, 2012/0028301, 2011/0183855, and 2009/0075378, each of which are incorporated herein by reference.

Screening of the libraries can be accomplished by various techniques known in the art. For example, PD-1 can be immobilized onto solid supports, columns, pins, or cellulose/poly(vinylidene fluoride) membranes/other filters, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads or used in any other method for panning display libraries.

For review of in vitro affinity maturation methods, see, e.g., Hoogenboom, 2005, Nature Biotechnology 23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51; and references therein.

4.3.2.2 Modifications of Anti-PD-1 Antibodies

Covalent modifications of anti-PD-1 antibodies are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of an anti-PD-1 antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the anti-PD-1 antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Other types of covalent modification of the anti-PD-1 antibody included within the scope of this present disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501; and Walsh, 2010, Drug Discov. Today 15:773-80), and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth, for example, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

An anti-PD-1 antibody of the present disclosure may also be modified to form chimeric molecules comprising an anti-PD-1 antibody fused to another, heterologous polypeptide or amino acid sequence, for example, an epitope tag (see, e.g., Terpe, 2003, Appl. Microbiol. Biotechnol. 60:523-33) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

Also provided herein are fusion proteins comprising an antibody provided herein that binds to a PD-1 antigen and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is fused is useful for targeting the antibody to cells having cell surface-expressed PD-1.

Also provided herein are panels of antibodies that bind to a PD-1 antigen. In specific embodiments, the panels of antibodies have different association rates, different dissociation rates, different affinities for a PD-1 antigen, and/or different specificities for a PD-1 antigen. In some embodiments, the panels comprise or consist of about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 antibodies or more. Panels of antibodies can be used, for example, in 96-well or 384-well plates, for assays such as ELISAs.

4.3.3 Preparation of Anti-PD-1 Antibodies

Anti-PD-1 antibodies may be produced by culturing cells transformed or transfected with a vector containing anti-PD-1 antibody-encoding nucleic acids. Polynucleotide sequences encoding polypeptide components of the antibody of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridomas cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expressing antibodies of the present disclosure include prokaryotes such as Archaebacteria and Eubacteria, including Gram-negative or Gram-positive organisms, eukaryotic microbes such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods as known in the art.

Methods for antibody production including vector construction, expression, and purification are further described in Plückthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (McCafferty et al. eds., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009); Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherischia coli, in Antibody Expression and Production (Al-Rubeai ed., 2011); and Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009).

It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-PD-1 antibodies. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis (1969); and Merrifield, 1963, J. Am. Chem. Soc. 85:2149-54). In vitro protein synthesis may be performed using manual techniques or by automation. Various portions of the anti-PD-1 antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-PD-1 antibody. Alternatively, antibodies may be purified from cells or bodily fluids, such as milk, of a transgenic animal engineered to express the antibody, as disclosed, for example, in U.S. Pat. Nos. 5,545,807 and 5,827,690.

4.3.4 Immunoconjugates

The present disclosure also provides conjugates comprising any one of the anti-PD-1 antibodies of the present disclosure covalently bound by a synthetic linker to one or more non-antibody agents.

In some embodiments, antibodies provided herein are conjugated or recombinantly fused, e.g., to a diagnostic or detectable molecule. The conjugated or recombinantly fused antibodies can be useful, for example, for monitoring or prognosing the onset, development, progression, and/or severity of a PD-1-mediated disease.

Such diagnosis and detection can be accomplished, for example, by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin or avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent material, such as, but not limited to, an acridinium based compound or a HALOTAG; radioactive materials, such as, but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga and ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, or ¹¹⁷Sn; positron emitting metals using various positron emission tomographies; and non-radioactive paramagnetic metal ions.

Also described herein are antibodies that are recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to generate fusion proteins, as well as uses thereof. In particular, provided herein are fusion proteins comprising an antigen-binding fragment of an antibody provided herein (e.g., a Fab fragment, Fc fragment, Fv fragment, F(ab)₂ fragment, a VH domain, a VH CDR, a VL domain, or a VL CDR) and a heterologous protein, polypeptide, or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide that the antibody is fused to is useful for targeting the antibody to a particular cell type, such as a cell that expresses PD-1. For example, an antibody that binds to a cell surface receptor expressed by a particular cell type may be fused or conjugated to a modified antibody provided herein.

Moreover, antibodies provided herein can be fused to marker or “tag” sequences, such as a peptide, to facilitate purification. In specific embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available. For example, as described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the “FLAG” tag.

Methods for fusing or conjugating moieties (including polypeptides) to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (Baldwin et al. eds., 1985); Thorpe et al., 1982, Immunol. Rev. 62:119-58; U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,723,125; 5,783,181; 5,908,626; 5,844,095; and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 10535-39; Traunecker et al., 1988, Nature, 331:84-86; Zheng et al., 1995, J. Immunol. 154:5590-600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-41).

Fusion proteins may be generated, for example, through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of anti-PD-1 antibodies as provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and U.S. Pat. No. 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). Antibodies, or the encoded antibodies, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. A polynucleotide encoding an antibody provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

An antibody provided herein can also be conjugated to a second antibody to form an antibody heteroconjugate as described, for example, in U.S. Pat. No. 4,676,980.

Antibodies that bind to PD-1 as provided herein may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

The linker may be a “cleavable linker” facilitating release of the conjugated agent in the cell, but non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include, without limitation, acid labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., Chari et al., 1992, Cancer Res. 52:127-31; and U.S. Pat. No. 5,208,020), thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance (see, e.g., Kovtun et al., 2010, Cancer Res. 70:2528-37).

Conjugates of the antibody and agent may be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further contemplates that conjugates of antibodies and agents may be prepared using any suitable methods as disclosed in the art (see, e.g., Bioconiugate Techniques (Hermanson ed., 2d ed. 2008)).

Conventional conjugation strategies for antibodies and agents have been based on random conjugation chemistries involving the ε-amino group of Lys residues or the thiol group of Cys residues, which results in heterogenous conjugates. Recently developed techniques allow site-specific conjugation to antibodies, resulting in homogeneous loading and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics. These include engineering of “thiomabs” comprising cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another method, selenocysteine is cotranslationally inserted into an antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry 48(50):12047-57).

4.4 Methods of Using the Antibodies and Compositions Thereof

Provided herein are methods of treating an immune disorder in a subject. In certain embodiments, the method comprises administering a PD-1 binding protein provided herein to the subject in an amount effective for treating the immune disorder. Also provided herein are methods of managing an immune disorder in a subject. In certain embodiments, the method comprises administering a PD-1 binding protein provided herein to the subject in an amount effective for managing the immune disorder. Also provided herein are methods of preventing an immune disorder in a subject. In certain embodiments, the method comprises administering a PD-1 binding protein provided herein to the subject in an amount effective for preventing the immune disorder. In some embodiments, the subject has an immune disorder. In other embodiments, the subject is at risk of having an immune disorder. In one embodiment, the subject is a subject in need thereof. In a specific embodiment, the PD-1 binding protein is a PD-1 antibody provided herein. In certain embodiments, the PD-1 binding protein is an antigen binding fragment of a PD-1 antibody provided herein.

In some embodiments of the various methods provided herein, the immune disorder is an inflammatory disease. In one embodiment, the inflammatory disease is uveitis. In specific embodiments of the various methods provided herein, the immune disorder is an autoimmune disease, such as rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, multiple sclerosis, lupus, ankylosing spondylitis, type I diabetes, Sjögren's syndrome, ulcerative colitis, neuromyelitis optica, celiac disease, scleroderma, and temporal arteritis. In some embodiments, the lupus is SLE, CLE, or lupus nephritis. In other embodiments of the various methods provided herein, the immune disorder is a hypersensitivity disease, including, for example, atopic dermatitis, hypersensitivity vasculitis, and allergy, e.g., allergic asthma, allergic rhinitis (hay fever), urticaria (hives), and anaphylaxis. In certain embodiments, the hypersensitivy disease is a T cell hypersensitivity disease.

In some embodiments of the various methods provided herein, the immune disorder is an autoimmune disease. In one embodiment, the autoimmune disease is rheumatoid arthritis. In another embodiment, the autoimmune disease is Crohn's disease. In yet another embodiment, the autoimmune disease is psoriasis. In still another embodiment, the autoimmune disease is multiple sclerosis. In another embodiment, the autoimmune disease is ankylosing spondylitis. In yet another embodiment, the autoimmune disease is type I diabetes. In still another embodiment, the autoimmune disease is Sjögren's syndrome. In a further embodiment, the autoimmune disease is ulcerative colitis. In another embodiment, the autoimmune disease is neuromyelitis optica. In yet another embodiment, the autoimmune disease is celiac disease. In one embodiment, the autoimmune disease is temporal arteritis. In another embodiment, the autoimmune disease is scleroderma. In one embodiment, the autoimmune disease is lupus. In one embodiment, the autoimmune disease is SLE. In another embodiment, the autoimmune disease is CLE. In still another embodiment, the autoimmune disease is lupus nephritis.

In some embodiments, the immune disorder is a hypersensitivity disease. In certain embodiments, the hypersensitivy disease is a T cell hypersensitivity disease. In one embodiment, the immune disorder is atopic dermatitis. In another embodiment, the immune disorder is hypersensitivity vasculitis. In yet another embodiment, the immune disorder is allergy. In one embodiment, the immune disorder is allergic asthma. In another embodiment, the immune disorder is allergic rhinitis (hay fever). In yet another embodiment, the immune disorder is urticaria (hives). In certain embodiments, the immune disorder is anaphylaxis.

The term “T cell hypersensitivity,” when used in reference to a immune disorder of a subject, refers to a transient or chronic abnormally high level of T cell effector function. In certain embodiments, the T cell effector function comprises secretion of TH2 cytokines. Exemplary TH2 cytokines include but are not limited to IL-2, IL-4, IL-9, IL-13, IL-31, and TSLP. In one embodiment, the TH2 cytokine is 1-2. In another embodiment, the TH2 cytokine is IL-4. In yet another embodiment, the TH2 cytokine is IL-9. In still another embodiment, the TH2 cytokine is IL-13. In one embodiment, the TH2 cytokine is IL-31. In another embodiment, the TH2 cytokine is TSLP. In some embodiments, the subject has a transient or chronic abnormally high level of two, three, four, five, six, seven, eight, nine, or ten different TH2 cytokines. In some embodiments, the subject has a transient or chronic abnormally high level of two, three, four, five, or six cytokines selected from the group consisting of 1-2, IL-4, IL-9, IL-13, IL-31, and TSLP.

In some embodiments, the various methods provided herein comprise administering an antibody that binds to PD-1, including a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope. In certain embodiments, the antibodies bind to human and/or cyno PD-1. In other embodiments, the antibodies do not bind to rodent PD-1 (e.g., a mouse PD-1). In one embodiment, an antibody binds to human PD-1. In another embodiment, an antibody binds to cyno PD-1. In another embodiment, the antibody binds to human PD-1 and cyno PD-1. In some embodiments, the antibody binds to human PD-1 and does not bind to a rodent PD-1 (e.g., a mouse PD-1). In some embodiments, the antibody binds to cyno PD-1 and does not bind to a rodent PD-1 (e.g., a mouse PD-1). In some embodiments, the antibody binds to human PD-1, binds to a cyno PD-1, and does not bind to a rodent PD-1 (e.g., a mouse PD-1). In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L1 to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L2 to a PD-1 polypeptide. In some embodiments, the anti-PD-1 antibodies do not block the binding of PD-L1 or PD-L2 to a PD-1 polypeptide. In other embodiments, the anti-PD-1 antibodies are humanized antibodies (e.g., comprising human constant regions) that bind PD-1, including a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope. In certain embodiments, the anti-PD-1 antibody comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the murine monoclonal antibodies provided herein, such as an amino acid sequence depicted in Tables 1-6. Accordingly, in some embodiments, the isolated antibody or functional fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from: (a) the antibody PD1AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Tables 1-2.

In some embodiments, the various methods provided herein comprise administering an antibody that comprises or consists of six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-2. In some embodiments, the antibody can comprise fewer than six CDRs. In some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-2. In some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of the monoclonal antibody selected from the group consisting of: (a) the antibody PD AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, and (f) the antibody PD1AB-6, described herein. Accordingly, in some embodiments, the various methods provided herein comprise administering an antibody comprises or consists of one, two, three, four, or five CDRs of anyone of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-2.

In some embodiments, the various methods provided herein comprise administering an antibody comprising one or more (e.g., one, two, or three) VH CDRs listed in Table 2. In other embodiments, the antibodies comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 1. In yet other embodiments, the antibodies comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 2 and one or more VL CDRs listed in Table 1. Accordingly, in some embodiments, the antibodies comprise a VH CDR1 having an amino acid sequence of SEQ ID NO:4. In some embodiments, the antibodies comprise a VH CDR2 having an amino acid sequence of SEQ ID NO:5. In some embodiments, the antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO:6. In some embodiments, the antibodies comprise a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from any one of the VH CDR1, VH CDR2, VH CDR3 amino acid sequence(s) as depicted in Table 2. In some embodiments, the antibodies comprise a VL CDR1 having an amino acid sequence of any one of SEQ ID NOS: 1 and 7. In another embodiment, the antibodies comprise a VL CDR2 having an amino acid sequence of SEQ ID NO:2. In some embodiments, the antibodies comprise a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In some embodiments, the antibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from any one of the VL CDR1, VL CDR2, VL CDR3 amino acid sequences as depicted in Table 1.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO:1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In other embodiments, the antibody comprises a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6; and a VL region comprising: (1) a VL CDR1 having an amino acid of SEQ ID NOS:7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody comprises a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6. In other embodiments, the antibodies comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In some embodiments, the antibodies comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NOS: 7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3.

In some embodiments, the various methods provided herein comprise administering an antibody comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-2. In particular embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4) and a VL CDR1 (SEQ ID NOS:1 or 7). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4) and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4) and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5) and a VL CDR1 (SEQ ID NOS: 1 or 7). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5) and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5) and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR3 (SEQ ID NO:6) and a VL CDR1 (SEQ ID NOS:1 or 7). In other embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6) and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6) and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), and a VL CDR1 (SEQ ID NOS: 1 or 7). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), and a VL CDR3 (SEQ ID NOS:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR1 (SEQ ID NOS:1 or 7). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), and a VL CDR1 (SEQ ID NOS:1 or 7). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR1 (SEQ ID NOS: 1 or 7), and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In some embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR1 (SEQ ID NOS:1 or 7). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS: 1 or 7), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS: 1 or 7), and a VL CDR2 (SEQ ID NO:2). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR2 (SEQ ID NO:2). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS: 1 or 7), and a VL CDR2 (SEQ ID NO:2). In some embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In some embodiments, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises a VH CDR1 (SEQ ID NO:4), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In one embodiment, the antibody comprises a VH CDR2 (SEQ ID NO:5), a VL CDR1 (SEQ ID NOS: 1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In other embodiments, the antibody comprises a VH CDR3 (SEQ ID NO:6), a VL CDR1 (SEQ ID NOS:1 or 7), a VL CDR2 (SEQ ID NO:2), and a VL CDR3 (SEQ ID NO:3). In another embodiment, the antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 1-2.

In some embodiments, the various methods provided herein comprise administering an antibody comprising CDRs disclosed herein that include consensus sequences derived from groups of related antibodies (see, e.g., Tables 1-2).

In other embodiments, the various methods provided herein comprise administering an antibody (or functional fragment thereof) that further comprises one, two, three, and/or four heavy chain FRs and/or one, two, three, and/or four light chain FRs from: (a) the antibody PD AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Tables 3-4.

In certain embodiments, the various methods provided herein comprise administering an antibody (or functional fragment thereof) that further comprises one, two, three, and/or four heavy chain FRs from: (a) the antibody PD1AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Table 4. In some embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-1. In some embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-2. In other embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-3. In certain embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-4. In other embodiments, the antibody heavy chain FR(s) is from the antibody PD1AB-5. In another embodiment, the antibody heavy chain FR(s) is from the antibody PD1AB-6.

In other embodiments, the various methods provided herein comprise administering an antibody (or functional fragment thereof) that further comprises one, two, three, and/or four light chain FRs from: (a) the antibody PD1AB-1, (b) the antibody PD1AB-2, (c) the antibody PD1AB-3, (d) the antibody PD1AB-4, (e) the antibody PD1AB-5, or (f) the antibody PD1AB-6, as shown in Table 3. In some embodiments, the antibody light chain FR(s) is from the antibody PD1AB-1. In some embodiments, the antibody light chain FR(s) is from the antibody PD1AB-2. In other embodiments, the antibody light chain FR(s) is from the antibody PD1AB-3. In certain embodiments, the antibody light chain FR(s) is from the antibody PD1AB-4. In other embodiments, the antibody light chain FR(s) is from the antibody PD1AB-5. In another embodiment, the antibody light chain FR(s) is from the antibody PD1AB-6.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH region that comprises, or further comprises: (1) a VH FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS:19 and 24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:21 and 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, the antibody comprises a VH region that comprises, or further comprises: (1) a VH FR1 having an amino acid of SEQ ID NO:19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, the antibody comprises a VH region that comprises, or further comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO: 19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO: 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, the antibody comprises a VH region that comprises, or further comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO: 24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In certain embodiments, the antibody comprises a VH region that comprises, or further comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO: 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22. In specific embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4.

Accordingly, in some embodiments, the various methods provided herein comprise administering a humanized antibody comprising a VH region that includes a VH FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS:19 and 24. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR1 having an amino acid sequence of SEQ ID NO:19. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR1 having an amino acid sequence of SEQ ID NO:24. In some embodiments, the humanized antibody comprises a VH region that includes a VH FR2 having an amino acid sequence of SEQ ID NO: 20. In some embodiments, the humanized antibody comprises a VH region that includes a VH FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:21 and 23. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR3 having an amino acid sequence of SEQ ID NO:21. In one embodiment, the humanized antibody comprises a VH region that includes a VH FR3 having an amino acid sequence of SEQ ID NO:23. In other embodiments, the humanized antibody comprises a VH region that includes a VH FR4 having an amino acid sequence of SEQ ID NO:22.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VL region that comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO:15; (3) a VL FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 16 and 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO:15; (3) a VL FR3 having an amino acid sequence of SEQ ID NOS: 16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In other embodiments, the VL region that comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO: 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17.

Accordingly, in some embodiments, the various methods provided herein comprise administering a humanized antibody that comprises a VL region that includes a VL FR1 having an amino acid sequence of SEQ ID NO: 14. In certain embodiments, the humanized antibody comprises a VL region that includes a VL FR2 having an amino acid sequence of SEQ ID NO: 15. In other embodiments, the humanized antibody comprises a VL region that includes a VL FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 16 and 18. In one embodiment, the humanized antibody comprises a VL region that includes a VL FR3 having an amino acid sequence of SEQ ID NOS: 16. In other embodiments, the humanized antibody comprises a VL region that includes a VL FR3 having an amino acid sequence of SEQ ID NO: 18. In yet other embodiments, the humanized antibody comprises a VL region that includes a VL FR4 having an amino acid sequence of SEQ ID NO: 17.

In other embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 19 and 24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:21 and 23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS:16 and 18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In certain embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO: 19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO: 19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In other embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO: 19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO: 19; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In certain embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:21; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3 and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3 and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In other embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:16; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

In certain embodiments, the various methods provided herein comprise administering an antibody comprising a VH region and a VL region, wherein the VH region comprises: (1) a VH FR1 having an amino acid sequence of SEQ ID NO:24; (2) a VH FR2 having an amino acid sequence of SEQ ID NO:20; (3) a VH FR3 having an amino acid sequence of SEQ ID NO:23; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NO:22; and wherein the VL region comprises: (1) a VL FR1 having an amino acid sequence of SEQ ID NO:14; (2) a VL FR2 having an amino acid sequence of SEQ ID NO: 15; (3) a VL FR3 having an amino acid sequence of SEQ ID NO:18; and/or (4) a VL FR4 having an amino acid sequence of SEQ ID NO:17. In some embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4. In other embodiments, the antibody comprises a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4. In yet other embodiments, the antibody comprises a VH region comprising all four of the above-referenced VH FR1, VH FR2, VH FR3, and VH FR4, and a VL region comprising all four of the above-referenced VL FR1, VL FR2, VL FR3, and VL FR4.

The methods provided herein, in certain embodiments, comprise administering an antibody comprising one or more (e.g., one, two, three, or four) VH FRs and one or more (e.g., one, two, three, or four) VL FRs listed in Tables 3-4. In particular, in some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24) and a VL FR1 (SEQ ID NO: 14). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24) and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24) and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR1 (SEQ ID NO: 14). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20) and a VL FR4 (SEQ ID NO: 17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR1 (SEQ ID NO:14). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR2 (SEQ ID NO:15). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NO:21) and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR1 (SEQ ID NO: 14). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22) and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR1 (SEQ ID NO: 14). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR1 (SEQ ID NO: 14). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR2 (SEQ ID NO: 15). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO: 15). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR2 (SEQ ID NO:15) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NO:19 or 24), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO: 15). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO: 17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15) and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO: 15). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15) and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15) and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15) and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR1 (SEQ ID NO:14). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO:14). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO: 15). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO: 14). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO: 14). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS: 16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NO:21), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO: 15). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NO:21), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO: 15). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR2 (SEQ ID NO: 15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR1 (SEQ ID NO:14). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR2 (SEQ ID NO: 15). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO: 15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR2 (SEQ ID NO:15). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR2 (SEQ ID NO:15). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR2 (SEQ ID NO: 15), a VL FR3 (SEQ ID NOS: 16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In some embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO: 15), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR2 (SEQ ID NO:20), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS: 19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In another embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR3 (SEQ ID NOS:16 or 18). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In one embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In another embodiment, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO: 17). In some embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR2 (SEQ ID NO:20), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In other embodiments, the antibody comprises a VH FR1 (SEQ ID NOS:19 or 24), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO:14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In one embodiment, the antibody comprises a VH FR2 (SEQ ID NO:20), a VH FR3 (SEQ ID NOS:21 or 23), a VH FR4 (SEQ ID NO:22), a VL FR1 (SEQ ID NO: 14), a VL FR2 (SEQ ID NO:15), a VL FR3 (SEQ ID NOS:16 or 18), and a VL FR4 (SEQ ID NO:17). In some embodiments, the antibody comprises any combination thereof of the VH FRs (SEQ ID NOS: 19-24) and the VL FRs (SEQ ID NOS:14-18) listed in Tables 3-4.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH region or VH domain. In other embodiments, the antibodies comprise a VL region or VL domain. In certain embodiments, the antibodies have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region. Exemplary VH regions, VH domains, VL regions and VL domains of antibodies useful in the methods are set forth elsewhere herein. In some embodiments, the antibodies have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region selected from the group consisting of SEQ ID NOS: 8-13 as set forth in Tables 5-6. In other embodiments, the antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6, as set forth in Tables 5-6.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO:4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO:5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO:6; and a VL region selected from the group consisting of SEQ ID NOS:8-10 as set forth in Table 5. In some embodiments, the VL region has an amino acid sequence of SEQ ID NO:8. In other embodiments, the VL region has an amino acid sequence of SEQ ID NO:9. In some embodiments, the VL region has an amino acid sequence of SEQ ID NO:10.

In other embodiments, the various methods provided herein comprise administering an antibody comprising a VH region selected from the group consisting of SEQ ID NOS: 11-13 as set forth in Table 6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 and 7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In other embodiments, the various methods provided herein comprise administering an antibody comprising a VH region selected from the group consisting of SEQ ID NOS:11-13 as set forth in Table 6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In yet other embodiments, the various methods provided herein comprise administering an antibody comprising a VH region selected from the group consisting of SEQ ID NOS: 11-13 as set forth in Table 6; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO:7; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO:2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO:3. In some embodiments, the VH region has an amino acid sequence of SEQ ID NO: 11. In some embodiments, the VH region has an amino acid sequence of SEQ ID NO:12. In some embodiments, the VH region has an amino acid sequence of SEQ ID NO:13.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a VH and a VL amino acid sequence of PD1AB-1. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 11, and a VL amino acid sequence of SEQ ID NO:8. In other embodiments, the antibody has a VH and a VL amino acid sequence of PD1AB-2. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 11, and a VL amino acid sequence of SEQ ID NO:9. In some embodiments, the antibody has a VH and a VL amino acid sequence of PD1AB-3. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 12, and a VL amino acid sequence of SEQ ID NO: 10. In other embodiments, the antibody has a VH and a VL amino acid sequence of PD1AB-4. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 12, and a VL amino acid sequence of SEQ ID NO:9. In some embodiments, the antibody has a VH and a VL amino acid sequence of PD1AB-5. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 13, and a VL amino acid sequence of SEQ ID NO:9. In other embodiments, the antibody has a VH and a VL amino acid sequence of PD1AB-6. In some embodiments, an antibody comprises a VH amino acid sequence of SEQ ID NO: 13, and a VL amino acid sequence of SEQ ID NO:8. In certain embodiments, the various methods provided herein comprise administering an antibody, which specifically binds to a PD-1 polypeptide (e.g., an ECD of PD-1, for example human PD-1), comprising a light chain and a heavy chain, wherein the light chain comprises a constant region having an amino acid sequence of SEQ ID NO:41. In other embodiments, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG1 Fc region having an amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the heavy chain does not comprise a human IgG1 Fc region having an amino acid sequence of SEQ ID NO:36. In certain embodiments, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG1-K322A Fc region having an amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG4 Fc region having an amino acid sequence of SEQ ID NO:38. In another embodiment, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG4P Fc region having an amino acid sequence of SEQ ID NO:39. In yet another embodiment the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises a human IgG4PE Fc region having an amino acid sequence of SEQ ID NO:40. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the heavy chain does not comprise a human IgG4PE Fc region having an amino acid sequence of SEQ ID NO:40. In still another embodiment, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises a constant region having an amino acid sequence of SEQ ID NO:41; and the heavy chain comprises an Fc region having an amino acid sequence selected from the group consisting of SEQ ID NOS:36-40.

In some embodiments, the various methods provided herein comprise administering an antibody comprising a light chain and a heavy chain, wherein the light chain comprises an amino acid sequence of SEQ ID NO:31. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO:32. In other embodiments, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO:33. In another embodiment, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO:34. In yet another embodiment, the antibody comprises a light chain and a heavy chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO:35. In one particular embodiment, the antibody comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:32. In another particular embodiment, the antibody comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:33. In yet another particular embodiment, the antibody comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:34. In still another particular embodiment, the antibody comprises a light chain and a heavy chain, wherein (i) the light chain comprises an amino acid sequence of SEQ ID NO:31; and (ii) the heavy chain comprises an amino acid sequence of SEQ ID NO:35.

In yet another embodiment, the various methods provided herein comprise administering an antibody that competes with one of the exemplified antibodies or functional fragments for binding to PD-1 provided herein. Such antibodies may also bind to the same epitope as one of the herein exemplified antibodies, or an overlapping epitope. Antibodies and fragments that compete with or bind to the same epitope as the exemplified antibodies are expected to show similar functional properties. The exemplified antigen-binding proteins and fragments include those with the VH and VL regions, and CDRs provided herein, including those in Tables 1-6. Thus, as a specific example, the antibodies useful in the methods provided herein include those that compete with an antibody comprising: (a) 1, 2, 3, 4, 5, or all 6 of the CDRs listed for an antibody listed in Tables 1-2; (b) a VH and a VL selected from the VH and the VL regions listed for an antibody listed in Tables 5-6; or (c) two light chains and two heavy chains comprising a VH and a VL as specified for an antibody listed in Tables 5-6. In some embodiments, the antibody is PD1AB-1. In some embodiments, the antibody is PD1AB-2. In some embodiments, the antibody is PD1AB-3. In some embodiments, the antibody is PD1AB-4. In some embodiments, the antibody is PD1AB-5. In some embodiments, the antibody is PD1AB-6.

Accordingly, provided herein is a method of treating an immune disorder in a subject, comprising administering to the subject an effective amount of an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is PD AB-1, PD AB-2, PD AB-3, PD AB-4, PD AB-5 or PD1AB-6. In some embodiments, the anti-PD-1 antibody is PD1AB-1. In some embodiments, the anti-PD-1 antibody is PD1AB-2. In some embodiments, the anti-PD-1 antibody is PD1AB-3. In some embodiments, the anti-PD-1 antibody is PD1AB-4. In some embodiments, the anti-PD-1 antibody is PD1AB-5. In some embodiments, the anti-PD-1 antibody is PD1AB-6. Also provided herein is a method of treating an immune disorder in a subject, comprising administering to the subject an effective amount of an antigen binding fragment of an anti-PD-1 antibody. In certain embodiments, the antigen binding fragment is a fragment of a PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 or PD1AB-6 antibody. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-1. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-2. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-3. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-4. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-5. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-6. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-1. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-1. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-1, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-1. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-2. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-2. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-2, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-2. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-3. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-3. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-3, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-3. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-4. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-4. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-4, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-4. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-5. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-5. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-5, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-5. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-6. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-6. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-6, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-6. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:8. In another embodiment, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:13. In yet another embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:8, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO: 13. In another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41. In yet another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:37. In still another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41 and a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:37. In one embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain comprising an amino acid sequence of SEQ ID NO:31. In another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:33. In yet another embodiment, the anti-PD-1 antibody, or antigen-binding fragment, thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO:31 and a heavy chain comprising an amino acid sequence of SEQ ID NO:33. In certain embodiments, the anti-PD-1 antibody comprises a human IgG1 constant region. In a specific embodiment, the IgG1 constant region comprises a K322A substitution. In one embodiment, the anti PD-1 antibody is PD1AB-6-K3 (PD1AB-6-K322A). In some embodiments, the subject has an immune disorder. In other embodiments, the subject is at risk of having an immune disorder. In certain embodiments of the methods provided herein, the method results in the alleviation of one or more symptoms of an immune disorder in a subject. In one embodiment, the subject is a subject in need thereof. In some embodiments, the immune disorder is an inflammatory disease. In one embodiment, the immune disorder is uveitis. In certain embodiments, the immune disorder is an autoimmune disease. In one embodiment, the immune disorder is rheumatoid arthritis. In another embodiment, the immune disorder is Crohn's disease. In yet another embodiment, the immune disorder is psoriasis. In still another embodiment, the immune disorder is psoriatic arthritis. In other embodiments, the immune disorder is multiple sclerosis. In another embodiment, the immune disorder is ankylosing spondylitis. In yet another embodiment, the immune disorder is type 1 diabetes mellitus. In still another embodiment, the immune disorder is Sjögren's syndrome. In one embodiment, the immune disorder, is ulcerative colitis. In another embodiment, the immune disorder is neuromyelitis optica. In yet another embodiment, the immune disorder is celiac disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the immune disorder is SLE. In another embodiment, the immune disorder is CLE. In still another embodiment, the immune disorder is lupus nephritis. In one embodiment, the immune disorder is temporal arteritis. In another embodiment, the immune disorder is scleroderma. In some embodiments, the immune disorder is a hypersensitivity disease. In certain embodiments, the hypersensitivy disease is a T cell hypersensitivity disease. In one embodiment, the immune disorder is atopic dermatitis. In another embodiment, the immune disorder is hypersensitivity vasculitis. In yet another embodiment, the immune disorder is allergy. In one embodiment, the immune disorder is allergic asthma. In another embodiment, the immune disorder is allergic rhinitis (hay fever). In yet another embodiment, the immune disorder is urticaria (hives). In certain embodiments, the immune disorder is anaphylaxis.

Also provided herein is a method of managing an immune disorder in a subject, comprising administering to the subject an effective amount of an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is PD AB-1, PD AB-2, PD AB-3, PD AB-4, PD AB-5 or PD1AB-6. In some embodiments, the anti-PD-1 antibody is PD1AB-1. In some embodiments, the anti-PD-1 antibody is PD1AB-2. In some embodiments, the anti-PD-1 antibody is PD1AB-3. In some embodiments, the anti-PD-1 antibody is PD1AB-4. In some embodiments, the anti-PD-1 antibody is PD1AB-5. In some embodiments, the anti-PD-1 antibody is PD1AB-6. Also provided herein is a method of managing an immune disorder in a subject, comprising administering to the subject an effective amount of an antigen binding fragment of an anti-PD-1 antibody. In certain embodiments, the antigen binding fragment is a fragment of a PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 or PD1AB-6 antibody. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-1. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-2. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-3. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-4. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-5. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-6. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-1. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-1. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-1, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-1. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-2. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-2. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-2, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-2. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-3. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-3. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-3, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-3. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-4. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-4. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-4, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-4. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-5. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-5. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-5, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-5. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-6. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-6. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-6, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-6. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:8. In another embodiment, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:13. In yet another embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:8, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO: 13. In another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41. In yet another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:37. In still another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41 and a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:37. In one embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain comprising an amino acid sequence of SEQ ID NO:31. In another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:33. In yet another embodiment, the anti-PD-1 antibody, or antigen-binding fragment, thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO:31 and a heavy chain comprising an amino acid sequence of SEQ ID NO:33. In certain embodiments, the anti-PD-1 antibody comprises a human IgG1 constant region. In a specific embodiment, the IgG1 constant region comprises a K322A substitution. In one embodiment, the anti PD-1 antibody is PD1AB-6-K3 (PD1AB-6-K322A). In some embodiments, the subject has an immune disorder. In other embodiments, the subject is at risk of having an immune disorder. In certain embodiments of the methods provided herein, the method results in the alleviation of one or more symptoms of an immune disorder in a subject. In one embodiment, the subject is a subject in need thereof. In some embodiments, the immune disorder is an inflammatory disease. In one embodiment, the immune disorder is uveitis. In certain embodiments, the immune disorder is an autoimmune disease. In one embodiment, the immune disorder is rheumatoid arthritis. In another embodiment, the immune disorder is Crohn's disease. In yet another embodiment, the immune disorder is psoriasis. In still another embodiment, the immune disorder is psoriatic arthritis. In other embodiments, the immune disorder is multiple sclerosis. In another embodiment, the immune disorder is ankylosing spondylitis. In yet another embodiment, the immune disorder is type 1 diabetes mellitus. In still another embodiment, the immune disorder is Sjögren's syndrome. In one embodiment, the immune disorder, is ulcerative colitis. In another embodiment, the immune disorder is neuromyelitis optica. In yet another embodiment, the immune disorder is celiac disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the immune disorder is SLE. In another embodiment, the immune disorder is CLE. In still another embodiment, the immune disorder is lupus nephritis. In one embodiment, the immune disorder is temporal arteritis. In another embodiment, the immune disorder is scleroderma. In some embodiments, the immune disorder is a hypersensitivity disease. In certain embodiments, the hypersensitivy disease is a T cell hypersensitivity disease. In one embodiment, the immune disorder is atopic dermatitis. In another embodiment, the immune disorder is hypersensitivity vasculitis. In yet another embodiment, the immune disorder is allergy. In one embodiment, the immune disorder is allergic asthma. In another embodiment, the immune disorder is allergic rhinitis (hay fever). In yet another embodiment, the immune disorder is urticaria (hives). In certain embodiments, the immune disorder is anaphylaxis.

Also provided herein is a method of preventing an immune disorder in a subject, comprising administering to the subject an effective amount of an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is PD AB-1, PD AB-2, PD AB-3, PD AB-4, PD AB-5 or PD1AB-6. In some embodiments, the anti-PD-1 antibody is PD1AB-1. In some embodiments, the anti-PD-1 antibody is PD1AB-2. In some embodiments, the anti-PD-1 antibody is PD1AB-3. In some embodiments, the anti-PD-1 antibody is PD1AB-4. In some embodiments, the anti-PD-1 antibody is PD1AB-5. In some embodiments, the anti-PD-1 antibody is PD1AB-6. Also provided herein is a method of preventing an immune disorder in a subject, comprising administering to the subject an effective amount of an antigen binding fragment of an anti-PD-1 antibody. In certain embodiments, the antigen binding fragment is a fragment of a PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5 or PD1AB-6 antibody. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-1. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-2. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-3. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-4. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-5. In some embodiments, the anti-PD-1 antibody fragment is a fragment of PD1AB-6. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-1. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-1. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-1, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-1. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-2. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-2. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-2, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-2. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-3. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-3. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-3, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-3. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-4. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-4. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-4, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-4. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-5. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-5. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-5, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-5. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-6. In some embodiments, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-6. In certain embodiments, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VL CDR1, VL CDR2, and VL CDR3 of PD1AB-6, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises VH CDR1, VH CDR2, and VH CDR3 of PD1AB-6. In one embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:8. In another embodiment, the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:13. In yet another embodiment, the VL of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO:8, and the VH of the anti-PD-1 antibody, or antigen binding fragment thereof, comprises an amino acid sequence of SEQ ID NO: 13. In another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41. In yet another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:37. In still another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41 and a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:37. In one embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a light chain comprising an amino acid sequence of SEQ ID NO:31. In another embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:33. In yet another embodiment, the anti-PD-1 antibody, or antigen-binding fragment, thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO:31 and a heavy chain comprising an amino acid sequence of SEQ ID NO:33. In certain embodiments, the anti-PD-1 antibody comprises a human IgG1 constant region. In a specific embodiment, the IgG1 constant region comprises a K322A substitution. In one embodiment, the anti PD-1 antibody is PD1AB-6-K3 (PD1AB-6-K322A). In some embodiments, the subject has an immune disorder. In other embodiments, the subject is at risk of having an immune disorder. In certain embodiments of the methods provided herein, the method results in the alleviation of one or more symptoms of an immune disorder in a subject. In one embodiment, the subject is a subject in need thereof. In some embodiments, the immune disorder is an inflammatory disease. In one embodiment, the immune disorder is uveitis. In certain embodiments, the immune disorder is an autoimmune disease. In one embodiment, the immune disorder is rheumatoid arthritis. In another embodiment, the immune disorder is Crohn's disease. In yet another embodiment, the immune disorder is psoriasis. In still another embodiment, the immune disorder is psoriatic arthritis. In other embodiments, the immune disorder is multiple sclerosis. In another embodiment, the immune disorder is ankylosing spondylitis. In yet another embodiment, the immune disorder is type 1 diabetes mellitus. In still another embodiment, the immune disorder is Sjögren's syndrome. In one embodiment, the immune disorder, is ulcerative colitis. In another embodiment, the immune disorder is neuromyelitis optica. In yet another embodiment, the immune disorder is celiac disease. In one embodiment, the autoimmune disease is lupus. In one embodiment, the immune disorder is SLE. In another embodiment, the immune disorder is CLE. In still another embodiment, the immune disorder is lupus nephritis. In one embodiment, the immune disorder is temporal arteritis. In another embodiment, the immune disorder is scleroderma. In some embodiments, the immune disorder is a hypersensitivity disease. In certain embodiments, the hypersensitivy disease is a T cell hypersensitivity disease. In one embodiment, the immune disorder is atopic dermatitis. In another embodiment, the immune disorder is hypersensitivity vasculitis. In yet another embodiment, the immune disorder is allergy. In one embodiment, the immune disorder is allergic asthma. In another embodiment, the immune disorder is allergic rhinitis (hay fever). In yet another embodiment, the immune disorder is urticaria (hives). In certain embodiments, the immune disorder is anaphylaxis.

In yet another embodiment, the various methods provided herein comprise administering an antibody or antigen-binding fragments thereof described herein that binds to a region, including an epitope, of human PD-1 or cyno PD-1. For example, in some embodiments, the antibody binds to a region of human PD-1 (SEQ ID NO:42) comprising amino acid residues 33 to 109 of human PD-1. In still another aspect, antibodies bind to a specific epitope of human PD-1.

In certain embodiments, the various methods provided herein comprise administering an antibody or antigen-binding fragment thereof that, when bound to PD-1, binds to at least one of residues 100-109 (SEQ ID NO:43) within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one of residues 100-105 (SEQ ID NO:44) within an amino acid sequence of SEQ ID NO:42. In particular embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one residue selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to at least one residue selected from the group consisting of L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to two or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In other embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to three or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to four or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In one embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to five or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to six or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In yet another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to seven or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In still another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to eight or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to nine or more residues selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In other embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to all ten residues from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42. In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to N33 within an amino acid sequence of SEQ ID NO:42. In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to T51 within an amino acid sequence of SEQ ID NO:42. In a particular embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to S57 within an amino acid sequence of SEQ ID NO:42. In one specific embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to L100 within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to N102 within an amino acid sequence of SEQ ID NO:42. In other embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to G103 within an amino acid sequence of SEQ ID NO:42. In another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to R104 within an amino acid sequence of SEQ ID NO:42. In yet another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to G103 and R104 within an amino acid sequence of SEQ ID NO:42. In still another embodiment, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to D105 within an amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to H107 within an amino acid sequence of SEQ ID NO:42. In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to PD-1, binds to S109 within an amino acid sequence of SEQ ID NO:42. Any combination of two, three, four, five, six, seven, eight, nine, ten or more of the above-referenced amino acid PD-1 binding sites is also contemplated.

In some embodiments of the various methods provided herein, the PD-1 binding protein (e.g., an anti-PD-1 antibody as described herein) is administered in an amount effective to treat T cell hypersensitivity. In some embodiments of the various methods provided herein, the PD-1 binding protein (e.g., an anti-PD-1 antibody as described herein) is administered in an amount effective to reduce undesirable or abnormally elevated levels of serum/plasma proinflammatory cytokines. In some embodiments of the various methods provided herein, the PD-1 binding protein (e.g., an anti-PD-1 antibody as described herein) is administered in an amount effective to activate PD-1 signaling in the subject.

In some embodiments of the various methods provided herein, the PD-1 binding protein (e.g., an anti-PD-1 antibody as described herein) is administered in an amount effective to downregulate PD-1 expression in the subject. In some embodiments of the various methods provided herein, the PD-1 binding protein (e.g., an anti-PD-1 antibody as described herein) is administered in an amount effective to attenuate T cell activity in the subject. In some embodiments, the effects of treatment are measured by (a) attenuating T cell activity; and/or (b) downregulating PD-1 expression. In certain embodiments, the effects of treatment are measured by attenuating T cell activity. In other embodiments, the effects of treatment are measured by downregulating PD-1 expression. In some embodiments, the effects of treatment are measured by (a) attenuating T cell activity; and (b) downregulating PD-1 expression. In certain embodiments, the attenuation of T cell activity is measured by inhibiting secretion of a cytokine. In some embodiments, the cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or a combination thereof. In one embodiment, the attenuation of T cell activity is measured by inhibiting secretion of IL-1. In some embodiments, the attenuation of T cell activity is measured by inhibiting secretion of 1-2. In another embodiment, the attenuation of T cell activity is measured by inhibiting secretion of IL-6. In yet another embodiment, the attenuation of T cell activity is measured by inhibiting secretion of IL-12. In still another embodiment, the attenuation of T cell activity is measured by inhibiting secretion of IL-17. In one embodiment, the attenuation of T cell activity is measured by inhibiting secretion of 1-22. In some embodiments, the attenuation of T cell activity is measured by inhibiting secretion of IL-23. In another embodiment, the attenuation of T cell activity is measured by inhibiting secretion of GM-CSF. In yet another embodiment, the attenuation of T cell activity is measured by inhibiting secretion of TNF-α. In still another embodiment, the attenuation of T cell activity is measured by inhibiting secretion of IFN-γ. In certain embodiments, the attenuation of T cell activity is measured by inhibition of T cell proliferation. In some embodiments, the inhibition of T cell proliferation is inhibition of CD4+ T cell proliferation. In other embodiments, the inhibition of T cell proliferation is inhibition of CD8+ T cell proliferation. In yet another embodiment, the attenuation of T cell activity is measured by downregulation of T cell activation markers. In one embodiment, the T cell activation marker is CD25. In another embodiment, the T cell activation marker is CD69. In still another embodiment, the attenuation of T cell activity is measured by upregulation of regulatory T cell biomarker. In one embodiment, the regulatory T cell biomarker is Foxp3. In still another embodiment, the attenuation of T cell activity is measured by increase of regulatory T cell numbers. In one embodiment, the regulatory T cell is induced Foxp3+ regulatory T cell. In one embodiment, the regulatory T cell is induced CD25+Foxp3+ regulatory T cell.

In certain embodiments, the attenuation of T cell activity is measured by upregulation of expression of an inhibitory receptor on the surface of T cells, wherein the inhibitory receptor is not PD-1. In some embodiments, the inhibitory receptor is selected from the group consisting of LAG-3, CTLA-4, and TIM-3, or a combination thereof. In one embodiment, the inhibitory receptor is LAG-3. In another embodiment, the inhibitory receptor is CTLA-4. In yet another embodiment, the inhibitory receptor is TIM-3.

In certain embodiments, the attenuation of T cell activity is measured by inhibition of T cell recall response. In one embodiment, the T cell recall response is measured by reduction of CD4+ T cell proliferation. In another embodiment, the T cell recall response is measured by inhibition of CD25 upregulation. In yet another embodiment, the T cell recall response is measured by reduction of CD4+ T cell proliferation and inhibition of CD25 upregulation.

In some embodiments, the attenuation of T cell activity is measured by increase of T cell exhaustion. In one embodiment, the increase of T cell exhaustion is measured by reduction of IL-7R^(hi) T cells. In another embodiment, the increase of T cell exhaustion is measured by increase of IL-7R^(lo) T cells. In yet another embodiment, the increase of T cell exhaustion is measured by reduction of IL-7R^(hi) T cells and increase of IL-7R^(lo) T cells. In one embodiment, the T cells are CD4+ T cells. In certain embodiments, the T cells are CD8+ T cells. In other embodiments, the T cells are CD4+ T cells and CD8+ T cells.

Moreover, an anti-PD-1 antibody or fragment thereof can be administered to a non-human mammal expressing PD-1 with which the antibody cross-reacts (e.g., a primate) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the present disclosure (e.g., testing of dosages and time courses of administration).

In certain embodiments of the various methods provided herein, a therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is administered to the subject. A therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is from about 0.005 to about 1,000 mg per day, from about 0.01 to about 500 mg per day, from about 0.01 to about 250 mg per day, from about 0.01 to about 100 mg per day, from about 0.1 to about 100 mg per day, from about 0.5 to about 100 mg per day, from about 1 to about 100 mg per day, from about 0.01 to about 50 mg per day, from about 0.1 to about 50 mg per day, from about 0.5 to about 50 mg per day, from about 1 to about 50 mg per day, from about 0.02 to about 25 mg per day, or from about 0.05 to about 10 mg per day.

In certain embodiments, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is about 0.1, about 0.2, about 0.5, about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, or about 150 mg per day.

In one embodiment, the recommended daily dose range of an anti-PD-1 antibody or antigen-binding fragment thereof for the conditions described herein lie within the range of from about 0.5 mg to about 50 mg per day, preferably given as a single once-a-day dose, or in divided doses throughout a day. In some embodiments, the dosage ranges from about 1 mg to about 50 mg per day. In other embodiments, the dosage ranges from about 0.5 to about 5 mg per day. Specific doses per day include 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg per day.

In a specific embodiment, the recommended starting dosage may be 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25 or 50 mg per day. In another embodiment, the recommended starting dosage may be 0.5, 1, 2, 3, 4, or 5 mg per day. The dose may be escalated to 15, 20, 25, 30, 35, 40, 45 and 50 mg/day.

In certain embodiments, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is from about 0.001 to about 100 mg/kg/day, from about 0.01 to about 50 mg/kg/day, from about 0.01 to about 25 mg/kg/day, from about 0.01 to about 10 mg/kg/day, from about 0.01 to about 9 mg/kg/day, from about 0.01 to about 8 mg/kg/day, from about 0.01 to about 7 mg/kg/day, from about 0.01 to about 6 mg/kg/day, from about 0.01 to about 5 mg/kg/day, from about 0.01 to about 4 mg/kg/day, from about 0.01 to about 3 mg/kg/day, from about 0.01 to about 2 mg/kg/day, or from about 0.01 to about 1 mg/kg/day.

The administered dose can also be expressed in units other than mg/kg/day. For example, doses for parenteral administration can be expressed as mg/m2/day. One of ordinary skill in the art would readily know how to convert doses from mg/kg/day to mg/m2/day to given either the height or weight of a subject or both (see, www.fda.gov/cder/cancer/animalframe.htm). For example, a dose of 1 mg/kg/day for a 65 kg human is approximately equal to 38 mg/m2/day.

In certain embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof administered is sufficient to provide a plasma concentration of the antibody at steady state, ranging from about 0.001 to about 500 M, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, from about 1 to about 50 μM, about 0.02 to about 25 μM, from about 0.05 to about 20 μM, from about 0.1 to about 20 μM, from about 0.5 to about 20 μM, or from about 1 to about 20 μM.

In other embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof administered is sufficient to provide a plasma concentration of the antibody at steady state, ranging from about 5 to about 100 nM, about 5 to about 50 nM, about 10 to about 100 nM, about 10 to about 50 nM or from about 50 to about 100 nM.

As used herein, the term “plasma concentration at steady state” is the concentration reached after a period of administration of a anti-PD-1 antibody or antigen-binding fragment thereof provided herein, e.g., PD1AB-6-K3. Once steady state is reached, there are minor peaks and troughs on the time dependent curve of the plasma concentration of the antibody.

In certain embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof administered is sufficient to provide a maximum plasma concentration (peak concentration) of the antibody, ranging from about 0.001 to about 500 μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, from about 1 to about 50 μM, about 0.02 to about 25 μM, from about 0.05 to about 20 μM, from about 0.1 to about 20 μM, from about 0.5 to about 20 μM, or from about 1 to about 20 μM.

In certain embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof administered is sufficient to provide a minimum plasma concentration (trough concentration) of the antibody, ranging from about 0.001 to about 500 μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, from about 1 to about 50 μM, about 0.01 to about 25 μM, from about 0.01 to about 20 μM, from about 0.02 to about 20 μM, from about 0.02 to about 20 μM, or from about 0.01 to about 20 μM.

In certain embodiments, the amount of the anti-PD-1 antibody or antigen-binding fragment thereof administered is sufficient to provide an area under the curve (AUC) of the antibody, ranging from about 100 to about 100,000 ng*hr/mL, from about 1,000 to about 50,000 ng*hr/mL, from about 5,000 to about 25,000 ng*hr/mL, or from about 5,000 to about 10,000 ng*hr/mL.

The anti-PD-1 antibody or antigen-binding fragment thereof provided herein, e.g., PD1AB-6-K3, can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day), intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term “daily” is intended to mean that a therapeutic antibody, such as PD1AB-6-K3, is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic antibody, such as PD1AB-6-K3, is administered daily for an uninterrupted period of at least 10 days to 52 weeks. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the antibody, such as PD1AB-6-K3, is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. The term “cycling” as used herein is intended to mean that a therapeutic antibody, such as PD1AB-6-K3, is administered daily or continuously but with a rest period.

In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. In one embodiment, the antibody, such as PD1AB-6-K3, is administered once a day. In another embodiment, the antibody, such as PD1AB-6-K3, is administered twice a day. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered three times a day. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered four times a day.

In certain embodiments, the anti-PD-1 antibody, such as PD1AB-6-K3, is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. In certain embodiments, the antibody, such as PD1AB-6-K3, is administered once per day for one week, two weeks, three weeks, or four weeks. In one embodiment, the antibody, such as PD1AB-6-K3, is administered once per day for one week. In another embodiment, antibody, such as PD1AB-6-K3, is administered once per day for two weeks. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once per day for three weeks. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once per day for four weeks.

In some embodiments, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is from about 0.001 to about 50 mg/kg, from about 0.01 to about 50 mg/kg, from about 0.01 to about 25 mg/kg, from about 0.01 to about 10 mg/kg, from about 0.01 to about 9 mg/kg, from about 0.01 to about 8 mg/kg, from about 0.01 to about 7 mg/kg, from about 0.01 to about 6 mg/kg, from about 0.01 to about 5 mg/kg, from about 0.01 to about 4 mg/kg, from about 0.01 to about 3 mg/kg, from about 0.01 to about 2 mg/kg, or from about 0.01 to about 1 mg/kg. In certain embodiments, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is about 0.001, about 0.003, about 0.005, about 0.01, about 0.03, about 0.05, about 0.1, about 0.3, about 0.5, about 1, about 3, about 5, about 10, about 30, or about 50 mg/kg. In one embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 0.001 mg/kg. In one embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 0.01 mg/kg. In one embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 0.1 mg/kg. In another embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 0.3 mg/kg. In another embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 0.5 mg/kg. In yet another embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 1 mg/kg. In still another embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 3 mg/kg. In one embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 5 mg/kg. In another embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 10 mg/kg. In yet another embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 30 mg/kg. In yet another embodiment, the therapeutically or prophylactically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is 50 mg/kg.

In some embodiments, the frequency of administration is once every week, once every two weeks, once every three weeks, or once every four weeks. In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every week. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks.

In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every week at a dosage of 0.1 mg/kg. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every week at a dosage of 0.3 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every week at a dosage of 0.5 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every week at a dosage of 1 mg/kg. In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every week at a dosage of 3 mg/kg. In another embodiment, the antibody, such as PD AB-6-K3, is administered once every week at a dosage of 5 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every week at a dosage of 10 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every week at a dosage of 30 mg/kg.

In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 0.1 mg/kg. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 0.3 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 0.5 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 1 mg/kg. In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 3 mg/kg. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 5 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 10 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every two weeks at a dosage of 30 mg/kg.

In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 0.1 mg/kg. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 0.3 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 0.5 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 1 mg/kg. In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 3 mg/kg. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 5 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 10 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every three weeks at a dosage of 30 mg/kg.

In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 0.1 mg/kg. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 0.3 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 0.5 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 1 mg/kg. In one embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 3 mg/kg. In another embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 5 mg/kg. In yet another embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 10 mg/kg. In still another embodiment, the antibody, such as PD1AB-6-K3, is administered once every four weeks at a dosage of 30 mg/kg.

Depending on the disease to be treated and the subject's condition, the antibody provided herein, e.g., PD1AB-6-K3, may be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, continuous intravenous, intracistemal injection or infusion, subcutaneous injection, or implant), inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g., transdermal or local) routes of administration. In one embodiment, the administration route is subcutaneous. In another embodiment, the administration route is intravenous. In yet another embodiment, the administration route is intramuscular. In still another embodiment, the administration route is intraperitoneal. In one embodiment, the administration route is continuous intravenous. In another embodiment, the administration route is intracistemal injection or infusion. In yet another embodiment, the administration route is implant. In still another embodiment, the administration route is inhalation. In one embodiment, the administration route is nasal. In another embodiment, the administration route is rectal. In yet another embodiment, the administration route is sublingual. In still another embodiment, the administration route is transdermal. Any anti-PD-1 antibody provided herein, e.g., PD1AB-6-K3, may be formulated, alone or together, in suitable dosage unit with pharmaceutically acceptable excipients, carriers, adjuvants, and vehicles, appropriate for each route of administration.

In some embodiments of the various methods provided herein, the method further comprises administering a therapeutically effective amount of a second active agent or a support care therapy. Second active agents can be large molecules (e.g., proteins) or small molecules (e.g., synthetic inorganic, organometallic, or organic molecules). In some embodiments, the second active agents are small molecules that can alleviate adverse effects associated with the administration of an antibody provided herein. However, like some large molecules, many are believed to be capable of providing a synergistic effect when administered with (e.g., before, after or simultaneously) an antibody provided herein. Examples of small molecule second active agents include, but are not limited to, anti-cancer agents, anti-inflammatory agents, immunosuppressive agents, and steroids.

4.5 Pharmaceutical Compositions

In one aspect, the present disclosure further describes pharmaceutical compositions for use in the various methods provided herein comprising at least one anti-PD-1 antibody of the present disclosure. In some embodiments, a pharmaceutical composition comprises 1) an anti-PD-1 antibody, and 2) a pharmaceutically acceptable carrier.

Pharmaceutical compositions comprising an antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (see, e.g., Remington, Remington's Pharmaceutical Sciences (18th ed. 1980)) in the form of aqueous solutions or lyophilized or other dried forms.

The antibodies of the present disclosure may be formulated in any suitable form for delivery to a target cell/tissue, e.g., as microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10:146-61; Malik et al., 2007, Curr. Drug. Deliv. 4:141-51), as sustained release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

An antibody provided herein can also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed, for example, in Remington, supra.

Various compositions and delivery systems are known and can be used with an antibody that binds to PD-1 as described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of a nucleic acid as part of a retroviral or other vector, etc. In another embodiment, a composition can be provided as a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, e.g., Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321:569-74). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody that binds to PD-1 as described herein) or a composition of the invention (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., 1985, Science 228:190-92; During et al., 1989, Ann. Neurol. 25:351-56; Howard et al., 1989, J. Neurosurg. 71:105-12; U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; PCT Publication Nos. WO 99/15154 and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

In yet another embodiment, a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, 1990, Science 249:1527-33. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies that bind to PD-1 as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT publication Nos. WO 91/05548 and WO 96/20698, Ning et al., 1996, Radiotherapy & Oncology 39:179-89; Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described herein.

All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a peptide sequence” includes a plurality of such sequences and so forth.

As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.

In addition, reference to a range of 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In a further example, reference to a range of 25-250, 250-500, 500-1,000, 1,000-2,500, 2,500-5,000, 5,000-25,000, 25,000-50,000 includes any numerical value or range within or encompassing such values, e.g., 25, 26, 27, 28, 29 . . . 250, 251, 252, 253, 254 . . . 500, 501, 502, 503, 504 . . . , etc.

As also used herein a series of ranges are disclosed throughout this document. The use of a series of ranges include combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth.

For the sake of conciseness, certain abbreviations are used herein. One example is the single letter abbreviation to represent amino acid residues. The amino acids and their corresponding three letter and single letter abbreviations are as follows:

alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp (D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly (G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys (K) methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S) threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V)

The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims.

5. EXAMPLES

The examples in this section (i.e., Section 5) are offered by way of illustration, and not by way of limitation.

5.1 Example 1: Generation of Anti-PD-1 Antibodies

5.1.1 Generation of Anti-PD-1 Antibodies

The parental PD-1-IgG1 mAb was initially generated by mouse immunization methods using human PD-1 extracellular domain (ECD) antigen or CHO-hPD-1 transfected cells. Initial characterization of a hybridoma pool identified a subclone designated PD1 Sub 1 producing an anti-human PD-1, PD-L1 non-blocking and PD-L2 non-blocking antibody (data not shown) with K_(D)˜6 nM measured by Biacore® to soluble antigen. The mouse V_(H) and V_(L) genes from the PD1 Sub 1 hybridoma were sequenced and used for human CDR-grafting into human γ1 and κ constant regions of the most homologous human V_(H) and V_(L) framework genes (IgH1-f and Vκ4-1, respectively) utilizing the closest J regions (IgH J6 and IgK J2, respectively). For human germline HG1 V_(H) and V_(L) regions only, the mouse CDR3 segments were placed into the human V_(H) and V_(L) framework germline genes, IgH 1-f and Vκ 4-1, respectively.

Stable HEK-293c18 cell lines expressing either CDR-grafted or germline HG1 antibodies in Deciduous™ constructs with stable AID (Activation-Induced Deaminase) were generated for use in a SHM-XEL™ affinity maturation platform (AnaptysBio, San Diego, Calif.). In situ generation of genetic diversity in the antibody variable domain resulted in cells expressing higher affinity variants of the parental antibody. These were isolated by flow cytometry using monomeric or dimeric hPD-1. Multiple rounds of affinity purification and selection yielded 6 corridors and 45 clones. Sanger and deep sequencing of these clones, along with additional “in silico SHM” events, resulted in rounds of site directed mutagenesis incorporating enriching mutations into VH and VL CDRs. The highest affinity binding 12 muteins were further characterized biochemically, biophysically for binding kinetics to PD-1, and for binding to full length PD-1 expressed on the surface of CHO cells. Functional characterization of the purified antibodies was performed in two assays: PD-L1 competition for binding to cell surface PD-1 and inhibitory activity in IL-2 production from reactivation of activated human CD4+ T cells.

Based on these methods, anti-PD-1 antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 were generated, as shown in Table 9.

TABLE 9 Characterization of anti-PD-1 antibodies HC/LC PD-L1 Antibody TOPO CDR-grafted HG1 K_(D) (Biacore) Competition CD4⁺ T cell IL-2 ID Vector ID HC/LC mutations K_(D) (KinExA) (IC₅₀) inhibition (EC₅₀) PD1AB-1 3015/3017 parental/parental 5 nM (n = 4) >100 nM (n = 4) 22 ± 4 nM (n = 5) 575 pM (n = 2) PD1AB-2 3015/3193 parental/germline 5 nM (n = 2) >100 nM (n = 2) 27 ± 4 nM (n = 3) 425 pM (n = 1) PD1AB-3 3653/3646 D76N/S77N 7 nM (n = 2) >100 nM (n = 1) 21 nM (n = 1) 350 pM (n = 1) PD1AB-4 3653/3193 D76N/germline 6 nM (n = 2) >100 nM (n = 1) 23 nM (n = 1) 500 pM (n = 1) PD1AB-5 3650/3193 V24A/germline 6 nM (n = 2) >100 nM (n = 1) 15 nM (n = 1) 400 pM (n = 1) PD1AB-6 3650/3017 V24A/parental 6 nM (n = 2) >100 nM (n = 1) 16 ± 3 nM (n = 3) 450 pM (n = 1)

5.1.2 CD4+ Reactivation Assay in Human PBMCs or Human Whole Blood

PD-1 expression was induced on human PBMCs isolated from leukocyte reduction system (LRS) with PHA activation 48 hours at 37° C. CD4+ T cells were purified from the PBMCs using CD4 isolation kits (Miltenyi Biotec, San Diego, Calif.) and replated onto 96-wells with immobilized anti-CD3 or anti-CD3 plus titrated anti-PD-1 or hIgG1 isotype control antibodies. Supernatants were collected at 24 and 48 hours for IL-2, IFN-γ, and IL-17 cytokine determinations. All six anti-PD-1 clones showed similar inhibition EC₅₀s, ranging from 20-36 nM for IL-2, 34-58 nM for IFN-γ, and 27-41 nM for IL-17, as shown in Table 10.

TABLE 10 Comparison of T cell attenuating activity of 6 lead antibodies in PBMC reactivation assay nM PD1AB-1 PD1AB-3 PD1AB-2 PD1AB-4 PD1AB-5 PD1AB-6 hIgG1 IL-2 EC₅₀ 21 ± 9 24 ± 5 24 ± 8  20 ± 7  36 ± 11 24 ± 7  >133 EC₇₅ 40 ± 9 46 ± 9 42 ± 9  39 ± 8  58 ± 18 33 ± 5  nM (n = 6) (n = 2) (n = 6) (n = 6) (n = 2) (n = 6) IFN-γ EC₅₀  46 ± 21 ND 58 ± 14 34 ± 21 ND 36 ± 22 >133 EC₇₅  85 ± 31 91 ± 29 64 ± 29 67 ± 30 nM (n = 3) (n = 3) (n = 3) (n = 3) IL-17 EC₅₀ 41 ± 9 ND 36 ± 12 27 ± 11 ND 37 ± 11 >133 EC₇₅ 65 ± 9 59 ± 10 52 ± 11 56 ± 13 nM (n = 5) (n = 5) (n = 5) (n = 5)

Inhibition of specific T cell function was then assessed in a human whole-blood matrix. Freshly drawn and heparinized human blood was plated onto wells immobilized with either anti-CD3 or anti-CD3 plus titrated anti-PD-1 or hIgG1 isotype control antibodies. Collected plasma at 24 and 48 hours were measured for IL-2 (24 hours) and IFN-γ/IL-17 (48 hours). Compared to three other tested antibody clones, PD1AB-6 showed a 2-3 fold increased potency in specific IL-2 (EC₅₀ 4.0+0.9 nM, n=4), IFN-γ (EC₅₀ 4.1+2.2 nM, n=2), and IL-17 (EC₅₀ 3.6+1.2 nM, n=3) inhibition, as shown in Table 11.

TABLE 11 Comparison of T cell attenuating activity of 4 lead molecules in whole blood assay EC₅₀ nM PD1AB-1 PD1AB-2 PD1AB-4 PD1AB-6 hIgG1 IL-2 Hu (n = 4) 8.3 ± 2.7 8.4 ± 1.1 10.2 ± 2.0  4.0 ± 0.9 >133 hIFN-γ Hu (n = 2) 5.9 ± 0.8 7.6 ± 0.9 9.2 ± 1.3 4.1 ± 2.2 >133 hIL-17 Hu (n = 3) 7.2 ± 1.3 8.9 ± 2.3 9.9 ± 3.7 3.6 ± 1.2 >133

5.1.3 Cell-Based Ligand Binding Assay

To assess ligand competition, a cell binding assay was put in-place to evaluate the identified six antibody clones. Briefly, individual antibody clones at semi-log concentrations from 100 nM to 100 pM were pre-mixed with 10 nM DyL650-PD-L1 before adding to human PD-1-CHO cells (2×10⁵ cells) for 45 minutes on ice. Cells were then washed before DyL650-PD-L1 binding was analyzed on a BD FACSArray™ and median fluorescence intensity relative to isotype control antibody was plotted at each concentration. As shown in FIGS. 1A-1B, PD1AB-6, as well as the other five clones, including the parental clone PD1AB-1, showed no significant competition against DyL650-PD-L1 binding up to 100 nM. In contrast, MDX 4H1 (AnaptysBio, San Diego, Calif.), an antagonist, ligand-blocking, PD-1 antibody dose-dependently blocked labeled PD-L1 binding, generating a binding EC₅₀˜5-10 nM.

5.1.5 Epitope Mapping

The PD-1 epitope was determined by solving the crystal structure of the PD1AB-6 Fab in complex with the human PD-1 extracellular domain to 1.8 Å resolution. The PD-1: PD1AB-6 Fab interaction site occurs on a distal side of PD-1 relative to the PD-1:PD-L1 interaction site (FIG. 2), consistent with the observation that PD-L1 and PD1AB-6 do not compete for PD-1 binding. PD1AB-6 Fab binds against a PD-1 β sheet, with substantial interactions formed with a PD-1 loop composed of residues 100-105 (FIG. 3). R104 on PD-1 engages multiple polar interactions with residues on the Fab CDR H1. The adjacent residue G103 also makes a tight polar interaction with the Fab. Both R104 and G103 are mutated in mouse PD-1 (to a histidine and arginine, respectively), providing a structural rationale for the lack of binding of PD1AB-6 to murine PD-1. The PD1AB-6 Fab regions that interact with PD-1 are the CDR H1, H2, H3, L1 and L2. Atomic details of the PD-1:PD1AB-6 Fab interactions are described in the Table 12. HC and LC residues that interact with PD-1 epitope are described. Abbreviations are as follows: HB-hydrogen bond, HYD-hydrophobic interaction, ION-ionic interaction.

TABLE 12 Atomic details of PD1AB-6:PD-1 interaction Type Chain Pos Chain Pos HB PD-1 ASN33.Nδ2 H ALA71.O HB PD-1 THR51.Oγ1 L SER50.O HB PD-1 SER57.Oγ H ASP69.Oδ2 HYD PD-1 LEU100.Cδ1 L PHE55.Cζ HYD PD-1 LEU100.Cδ1 L LEU115.CD1 HB PD-1 ASN102.Nδ2 L TYR114.O HB PD-1 ASN102.Oδ1 L SER117.N HB PD-1 ASN102.O H TYR50.Oη HB PD-1 GLY103.O H TYR126.Oη HB PD-1 ARG104.Nη2 H LYS47.O HB PD-1 ARG104.Nη1 H ASP48.O HB PD-1 ARG104.Nη2 H ASP69.Oζ1 ION PD-1 ARG104.Nη2 H ASP69.Oζ1 HB PD-1 ASP105.Oδ1 H SER125.Oγ HB PD-1 HIS 107.Nδ1 L SER50.Oγ HB PD-1 SER109.Oγ L SER50.O

5.1.6 Generation of Variants of PD1AB-6

PD1AB-6 IgG1 antibody (PD1AB-6-IgG1) and Fc modified IgG4PE antibody (PD1AB-6-4PE) were generated. PD1AB-6-4PE was designed to have significantly lower Fc-mediated effector function. The CH region, γ4 contains two non-standard amino acids substitutions, S228P and L235E (EU numbering systems, Kabat and Wu 1991). Serine 228, a common amino acid type in the hinge of IgG4, was changed to proline, a less commonly observed amino acid type in IgG4 and highly conserved amino acid in IgG1. This change significantly reduced the level of “half-antibody” that is commonly observed in the production of IgG4-subclass antibody. Leucine 235, one of the critical amino acids involved in heavy chain interactions with Fcγ receptors was changed to glutamic acid. The L235E substitution significantly reduced the interaction of γ4 chain to FcγR, eliminating ADCC and Fc-receptor-mediated elimination of PD-1-expressing normal cells. In addition, inherent lack of complement binding by γ4 heavy chain renders the PD1AB-6-4PE molecule devoid of CDC function. Two other variants were generated to minimize binding affinity to Clq for reduced CDC (FIG. 4). To generate PD1AB-6-K3, lysine 322 was substituted with alanine in PD1AB-6-IgG1. The K322A substitution is reported to suppress Clq binding on rituximab, a chimeric antibody with a human IgG1 Fc (Idusogie et al., 2000, J. Immunol. 164(8):4178-84). PD1AB-6-4P was generated by converting the Fc-backbone of the PD1AB-6-IgG1 to the Fc-backbone of IgG4 with S228P substitution. Serine 228, a common amino acid type in the hinge of IgG4, was changed to proline, a less commonly observed amino acid type in IgG4 and highly conserved amino acid in IgG1. This change significantly reduces the level of half-antibody that is frequently observed in the production of IgG4-subclass antibody. IgG4 antibody was reported to have attenuated ADCC and CDC function (Overdijk et al., 2012, J. Immunol. 189(7):3430-38). All changes were created in the CH region with no changes in the variable regions. The amino acid sequences of the heavy and light chains of PD1AB-6-IgG1 are labeled LC_PD1AB-6-IgG1 and HC_PD1AB-6-IgG1, respectively (FIG. 4). The two heavy chain variants include HC_PD1AB-6-IgG1-K322A and HC_PD1AB-6-IgG4P. The light chain LC_PD1AB-6-IgG1 is paired with the three individual heavy chains to generate PD1AB-6-IgG1, PD1AB-6-K3, and PD1AB-6-4P, respectively.

5.1.8 Cell Line Development and Antibody Manufacturing from Transient Transfection

5.1.8.1 Molecular Cloning of the Heavy and Light Chains

IgG LC expression vector pFUSE2ss-CLIg-hk and IgG HC expression vector pFUSEss-CHIg-hG1 were purchased from InvivoGen (San Diego, Calif.).

The amino acid sequence encoding LC_PD1AB-6-IgG1 (FIG. 4) was converted into a codon-optimized gene sequence for protein expression in mammalian cells. Restriction enzyme sites EcoRI at the 5′-end and NheI at the 3′-end were added to the optimized gene. The optimized LC gene with EcoRI and NheI sites were synthesized producing an insert fragment. The IgG LC expression vector pFUSE2ss-CLIg-hk was digested with EcoRI and NheI producing approximately a 3.5 kb pFUSE2ss-CLIg-hk-EcoRI/NheI fragment. The insert fragment was ligated into pFUSE2ss-CLIg-hk-EcoRI/NheI fragment resulting in production of pJS-1 which is pFUSE2ss-CLIg-hk-LC_PD1 AB-6-IgG1.

The amino acid sequence encoding HC_PD1AB-6-IgG1, HC_PD1AB-6-IgG1-K322A, or HC_PD1AB-6-IgG4P (FIG. 4) was converted into a codon-optimized gene sequence for protein expression in mammalian cells. Restriction enzyme sites EcoRI at the 5′-end, a constant region from a stop codon (after the 3′-end of HC sequence in pFUSEss-CHIg-hG1) to HpaI at the 3′-end were added. The optimized genes with EcoRI and HpaI sites were synthesized producing insert fragments containing the genes encoding HC_PD1AB-6-IgG1, HC_PD1AB-6-IgG1-K322A, and HC_PD1AB-6-IgG4P, respectively. The IgG HC expression vector pFUSEss-CHIg-hG1 was digested with EcoRI and HpaI producing approximately a 3.4 kb pFUSEss-CHIg-hG1-EcoRI/HpaI fragment. The insert fragments were ligated into pFUSEss-CHIg-hG1-EcoRI/HpaI fragment resulting in production of pJS-2, pJS-3, and pJS-12, which are pFUSEss-CHIg-hG1-HC_PD1AB-6-IgG1, pFUSEss-CHIg-hG1-HC_PD1AB-6-IgG1-K322A, and pFUSEss-CHIg-hG1-HC_PD1AB-6-IgG4P, respectively.

5.1.8.2 Protein Production

All three variants of PD1AB-6-IgG1, PD1AB-6-K3, and PD1AB-6-4P were manufactured at the laboratory scale in shake-flasks for in vitro and in vivo efficacy studies. PD1AB-6-4P and PD1AB-6-K3 antibodies for non-GLP toxicology studies and additional characterization were manufactured in 50 L bioreactors (50 L stirred tanks and 50 L wave bags) using FreeStyle™ MAX CHO expression system as well as Expi293™ expression system from Life Technologies (Carlsbad, Calif.). FreeStyle™ MAX CHO expression system was used for transient transfection of CHO-S cells using manufacturer's standard protocol. Expi293™ expression system was used for transient transfection of Expi293 cells using manufacturer's standard protocol. A 3:2 ratio of light chain versus heavy chain was used for DNA mixture at 1 mg per 1 L of culture during the transfection. Cells were seeded at 0.5 million cells/mL in a 50 L bioreactor at 37° C. and grew over night to reach 1 million cell/mL. Cells then were transfected using manufacturer's standard protocols. On day one post-transfection, 1 mM sodium butyrate plus 1% v/v of feed media (Yeastolate, CHO CD EfficientFeed™ A, Glutamax and Glucose) was added to bioreactor, and temperature was dropped to 32° C. Forty liters of cells plus additives were seeded for a 50 L stirred tank, and 25 L of cells plus additives were seeded for a 50 L wave bioreactor. Cell viability and titer were monitored every day, and batches were harvested when cell viability dropped below 50%. Vi-Cell™ instrument was used for viability analysis, and Octet RED equipped with Anti-Human IgG sensor was used for titer analysis using purified antibody for the standard curve. Cells and supernatant were harvested using GE Life Sciences depth filtration and sterilization columns, ULTA Prime GF 5 μm capsules were used for depth filtration followed by ULTA Pure HC 0.6/0.2 μm sterilization capsules. Clarified supernatant were concentrated 5-8 fold using cross flow filtration, 50 Kd cut off Kvick™ Lab SCU from GE Life Science were used for TFF. The titer and maximum cell densities obtained for each isotype at harvest are given in Table 13.

TABLE 13 Productivity in Fed-batch Bioreactor (50 Liter) Isotype Cell Cell Titer Cell density at harvest pool Line/Volume (mg/l) (1 × 10⁶ cells/mL) PD1AB-6-4P Expi293/25 L 22 3.2 PD1AB-6-4P CHO-S/50 L 9 3 PD1AB-6-K3 Expi293/50 L 31 4.5 PD1AB-6-K3 CHO-S/50 L 15 2

5.1.8.3 Protein Purification

Purification of the materials produced was performed by a series of downstream purification steps including protein A affinity chromatography and low pH virus inactivation, followed by IEX interaction (Capto™ Adhere & Capto™ SP ImpRes) chromatography steps. The purified antibody is bulk formulated by buffer exchanged against (10 mM Succinate pH 5.5, 9% sucrose, 0.05% PS20) buffer, filtered through 0.2 m filter, and aliquoted.

The protein A affinity chromatography was carried out with MabSelect SuRe™, designed to capture the product and to remove process related impurities. The subsequent virus inactivation step was performed under acidic conditions (pH 3.4±0.1 for 45 min.) followed by conditioning of the inactivation pool to pH 5.5±0.1. After virus inactivation, an anion exchanger was used in a flow-through mode for intermediate polishing step using Capto™ Adhere to remove impurities such as aggregates, DNA, host cell protein, and endotoxin. The product pool was conditioned to pH 6.5±0.1, and the conductivity was reduced to 2 mS/cm prior to the next process step. Cation exchanger Capto™ SP ImpRes was used as a polishing step, and the product was resolved at 10 mS/cm. The antibody was then buffer exchanged in stock solution (10 mM Succinate, 9% sucrose, 0.05% PS20, pH 5.5) and concentrated to 20 mg/mL. The product pool was then filtered through 0.2 m filter and aliquoted.

5.1.9 Cell-Based PD-1 Binding Assay

PD1AB-6-IgG1 binding was evaluated on CHO cells expressing human PD-1 and cyno PD-1 (FIGS. 5A-5B), and on primary human PBMC (FIG. 6) and cyno PBMC (FIG. 7).

CHO cells expressing human PD-1 and cyno PD-1 were incubated with various concentration of unlabeled PD1AB-6-IgG1 antibody for 30 minutes at 4° C., washed, and stained with anti-human IgG Fc (eBioscience, San Diego, Calif.) for 30 minute at 4° C. Human IgG1 Fc was used as a negative control. PD1AB-6-IgG1 binds to human PD-1 expressed on CHO cells with an EC₅₀=0.4 nM and binds to Cyno PD-1 expressed on CHO cells with an EC₅₀=0.8 nM (FIGS. 5A-5B).

Human PBMCs were activated with 1 μg/mL plate bound anti-CD3 for 3 days to induce PD-1 expression on T cells. Cells were incubated with various concentration of unlabeled PD1AB-6-IgG1 antibody for 30 minutes at 4° C., washed, and stained with anti-human IgG Fc (eBioscience, San Diego, Calif.) for 30 minute at 4° C. Human IgG1 Fc was used as a negative control. Geometric MFI was determined on CD4+ T cells. Data from 1 of 2 human healthy donors are shown in FIG. 6.

Cyno PBMCs were activated with 1 μg/mL anti-cyno CD3/CD28 for 2 days to induce PD-1 expression on T cells. Cells were incubated with various concentration of unlabeled PD1AB-6-IgG1 antibody for 30 minutes at 4° C., washed, and stained with anti-human IgG Fc (eBioscience) for 30 minute at 4° C. Human IgG1 Fc was used as a negative control. Geometric MFI was determined on CD4+ T cells. Data from 1 of 2 cyno donors are shown in FIG. 7.

5.1.10 Fc Receptor Binding Assay

To confirm the objectives of variants generation, i.e., decreased FcγR-mediated effector function, FcγR binding to the PD1AB-6-K3 and PD1AB-6-4P variants were analyzed by two methodologies. First, binding was tested with displacement FcγR assays using Cisbio Tag-Lite® detection (FIGS. 8A-8D). HEK293 cells engineered to express specific FcγRs (FcγRI, FcγRIIIa, or FcγRIIb) prelabeled with a terbium (Tb) donor dye were mixed with reference controls or PD1AB-6-IgG1, PD1AB-6-K3, and PD1AB-6-4P antibodies over log concentrations ranging from 10000 nM to 0.1 pM. A second, human-hIgG-d2 (acceptor) was then added to compete for receptor binding. Detection of a Fluorescence Resonance Energy Transfer (FRET) signal generated by Tb-d2 proximity is measured and is inversely proportional to PD1AB-6 variant-bound FcγR. As shown in FIGS. 8A-8D, the PD1AB-6-K3 variant showed decreased binding to FcγRIIIa (CD16), the low affinity receptor on NK cells responsible for ADCC activity. Binding to FcγRI (expressed on granulocytes, dendritic cells (DCs), or monocytes) was similar to parental PD1AB-6-IgG1 molecule.

Second, both PD1AB-6-K3 and PD1AB-6-4P variants were tested in FACS-based binding assays (FIGS. 9A-9C). Briefly, FcγRI-CHO or FcγRIIIaV158-CHO expressing cell lines were detached and washed prior to mixing with the PD1AB-6-K3 and PD1AB-6-4P variants over different concentrations for 1 hour on ice. PD1AB-6 variant-bound cells were detected with a labeled PE-conjugated F(ab′)₂ goat anti-human secondary antibody for an additional hour on ice, washed, and fixed prior to analysis by FACS, and mean fluorescence intensity was plotted at each concentration. The PD1AB-6-4P variant returned significantly higher binding EC₅₀s (>15× and >21X) against FcγRI and FcγRIIIa lines, respectively (FIGS. 9A-9C).

5.1.11 In Vitro ADCC Assay

The ability of PD1AB-6 variants to induce ADCC was evaluated in co-culture assay involving natural killer (NK) cells from healthy donors and PD-1 expressing target cells. Target cells (NCI-OCI-Ly3) pre-treated with PD1AB-6 variants were co-cultured with activated NK cells for 4 hours. Supernatant LDH concentration was used to calculate specific lysis. EC₅₀ (nM) was calculated using Prism. Error bar represents experimental triplicates. Data are 2 representatives of 4 individual healthy donors. As shown in FIGS. 10A-10B, titration of PD1AB-6-IgG1 induced dose dependent ADCC, while PD1AB-6-K3 showed reduced ADCC activity.

5.1.12 In Vitro CDC Assay

The ability of PD1AB-6 variants to induce CDC was evaluated using PD-1 expressing CD20⁺ NCI-OCI-Ly3 cells. Target cells (NCI-OCI-Ly3) pre-treated with antibodies were cultured in serum-free media supplemented with 5% rabbit complement for 4 hours. Cell lysis was determined by 7-AAD⁺ cells by FACS. Data are representative of 3 independent experiments: (i) CDC activity of PD1AB-6-IgG1 and anti-CD20 IgG1; (ii) CDC activity of PD1AB-6-IgG1 and PD1AB-6-K3; (iii) CDC activity of PD1AB-6-4P and commercial mouse anti-PD-1 IgG antibody. As shown in FIG. 11, PD1AB-6-K3 consistently did not induce CDC (n=3). Parental PD1AB-6-IgG1 and PD1AB-6-4P also did not induce CDC. This was not due to resistance of the target cell line to complement killing, since anti-CD20 IgG1 repeatedly induced dose dependent CDC on NCI-OCI-Ly3 cells in presence of 5% rabbit complement.

5.2 Example 2: Activity Assays

5.2.1 Human T Cell Activation Assay

Functional assessment of PD1AB-6 variants on inhibiting T cell effector function was performed by two methods. In one assay, the peripheral blood mononuclear cells were preactivated to express PD-1 and restimulated in the presence of soluble PD1AB-6-K3 (FIG. 12). Peripheral blood mononuclear cells (PBMCs), from healthy donors were preactivated with the mitogen, PHA, for 48 h to upregulate PD-1 expression. These cells were then restimulated using anti-CD3 conjugated Dynabeads® (Life Technologies, Carlsbad, Calif.) in the presence of diluted PD1AB-6 variants over a range of 100 nM to 0.1 nM final concentration. T cell activation was measured using IL-2 levels in culture supernatants at 24 h post-stimulation. As shown in FIG. 12, PD1AB-6-K3 and the two other variants showed potent T cell inhibitory activity in this assay with an EC₅₀ of 5-25 nM.

A second assay was used for direct ex vivo measurement of PD1AB-6-K3 in inhibiting T cell function (FIG. 13). This was done by directly plating fresh human whole blood in 96-well plates co-coated with anti-CD3+/−PD1AB-6-K3, and measuring IL-17 and IFN-γ levels as readouts of T cell activation. CTLA4Ig (Orencia®) was used as a positive control in these assays, and human IgG Fc fragment was used as a negative control. As shown in FIG. 13, overall, PD1AB-6-K3 trended towards better efficacy than PD1AB-6-4P. Negative control, hIgG Fc, showed no activity with an EC₅₀>100 nM.

5.2.2 Cynomolgus Monkey Crossreactivity Assay

Determination of functional cynomolgus monkey cross reactivity with lead PD1AB-6-K3 was performed similarly to human samples, using freshly isolated cynomolgus PBMCs, activated with anti-cyno CD3, CD28, and PD1AB-6 variants as indicated. CTLA4Ig was used as a positive control in these assays, and hIgG1 Fc was used as a negative control. Culture supernatants were removed after 48 hours for cytokine determinations using cynomolgus IL-2 MSD assays. As shown in Table 14, these assays demonstrated that PD1AB-6-K3 attenuated cyno T cells cytokine secretion to levels comparable to the positive control, CTLA4Ig, and the activity was comparable to that seen in human assays.

TABLE 14 PD1AB-6-K3 activity in cyno PBMC assay PD1AB-6-IgG1 PD1AB-6-K3 PD1AB-6-4P CTLA4Ig IL-2 EC50 (nM) NHP1 0.28 0.24 <0.1 Not done NHP2 4.9 2.5 1.24 Not done NHP3 N.D. 9.24 16.5 14 NHP4 N.D. 1.22 1.23 6

5.2.3 In Vitro Mechanism of Action

Several antibodies that bind to T cell surface molecules, such as CD3 and CD4, lead to signaling and subsequent downregulation of surface expression of those molecules. Since PD1AB-6 antibody is designed to provide an agonist signal via PD-1, it was of interest to evaluate PD-1 expression after PD1AB-6 treatment in vitro.

5.2.3.1 Decreased PD-1 Expression after PD1AB-6 Treatment

Human PBMC from different donors were activated with 1 μg/mL plate bound anti-CD3+0.25 μg/mL plate bound anti-CD28 with various concentration of soluble control IgG1 or PD1AB-6-IgG. After 4 hours to 72 hours incubation, cells were stained for CD3, CD45RO, and PD-1 to assess PD-1 expression on T cells. FIGS. 14A-14C show reduced expression of PD-1 on human CD3+ T cells after 48 hours PD1AB-6-IgG1 treatment. Analysis of PD-1 expression showed that PD1AB-6-IgG1 treatment led to downregulation of PD-1 expression on the surface of T cells (representative histogram from one donor in FIGS. 14A-14B and analysis of mean fluorescence intensity at 48 hours, across three donors and various antibody concentrations in FIG. 14C). PD-1 downregulation was seen as early as 4 hours of incubation with PD1AB-6-IgG1. Downregulation of surface PD-1 expression was likely due to PD1AB-6 induced signaling via PD-1, and was through similar mechanism to what is observed with T cell receptor signaling (San Jose et al., 2000, Immunity 12(2): 161-70).

5.3 Example 3: Physicochemical Characterization of PD1AB-6 Variants

5.3.1 Biacore® Binding Analysis

Purified PD1AB-6 variant antibodies were analyzed on Biacore® T200 for binding to hPD1 antigen using capture method. Fc-specific anti-human IgG was immobilized on Fc2, and Fc1 was left blank as reference channel. Purified PD1 antibodies were captured on anti-Human IgG, and internally produced hPD1 antigen (PDI_002) was flowed over both channels using two fold dilution series from 100 nM to 200 pM to determine kinetics of binding. The PD-1 used was the extracellular domain of human PD-1 (residues 32-160) expressed in E. Coli as inclusion bodies and refolded. Surface was regenerated between each antigen concentration using 3M Magnesium Chloride. Examples of binding kinetics as well as values of k_(on), k_(off), and K_(D) for PD1AB-6-IgG1, PD1AB-6-4P, and PD1AB-6-K3 are shown in FIGS. 15A-15C. All three variants had similar rates of association and dissociation to the PD-1 antigen with comparable K_(D) values of 19-22 nM.

5.4 Example 4: In Vitro Pharmacology

5.4.1 Tetanus Toxoid Antigen (TTX) Recall Assay

The effect of PD1AB-6 on antigen specific CD4+ T cell function was tested using soluble PD1AB-6 in a TTX recall assay conducted with PBMCs from healthy donors (AllCells, LLC, Alameda, Calif.; San Diego Blood Bank, San Diego, Calif.; Astarte Biologics LLC, Redmond, Wash.). PBMCs were pre-incubated with PD1AB-6 for 1 hour before stimulation with 1 μg/mL of tetanus toxoid clostridium TE (EMD Millipore, San Diego, Calif. 58223125UG) to induce tetanus-specific CD4+ T cell activation. After 7 days of TTX incubation, PBMCs were evaluated for CD4+ T cell proliferation as measured by cell division indicated by CellTrace™ Violet low cells by flow cytometry. LIVE/DEAD® dye (LIVE/DEAD® Fixable Blue Dead Cell Stain, Molecular Probes™ L23105) was used to exclude dead cells from the analysis. In all three donors, PD1AB-6 inhibited CD4 recall response with an IC₅₀ of 0.01-0.02 μg/mL (FIG. 16).

In a separate experiment, PBMCs pre-treated with PD1AB-6-K3 were incubated with TTX for seven days at various serum concentrations and evaluated for antigen-dependent proliferation and activation, as indicated by CD25 up-regulation. Healthy donor PBMCs were labeled with cell proliferation dye (CellTrace™ Violet, Thermo Fisher C34571) (37° C. for 20 minutes). CellTrace Violet dye-labeled PBMCs (5×10⁶ cells/mL) were pre-incubated with PD1AB-6-K3 or PD1AB-6-IgG1 at various concentrations (0.01 nM to 100 nM) for 1 hour before stimulation with 1 μg/mL of tetanus toxoid to induce tetanus-specific CD4+ T cell activation. An isotype matched anti-RSV IgG1-K322A was included as a negative control, while PD-L1 Fc was added as a treatment control to determine if activating the PD-1 pathway alone would inhibit T cells. PBMCs were stained with surface markers to identify CD4+/CD8-T cells, and T cell proliferation was measured by cell division detected by CellTrace™ Violet low cells by flow cytometry. LIVE/DEAD® dye (LIVE/DEAD® Fixable Blue Dead Cell Stain, Molecular Probes™ L23105) was used to exclude dead cells from the analysis. FACS analysis (BD FACS Canto flow cytometer, Flowjo® software (Tree Star, Inc)) indicated that all CD4+ cells undergoing tetanus toxoid-dependent proliferation were positive for CD25 expression and the majority expressed PD-1.

5.4.2 Ex Vivo Whole Blood Cytokine Secretion

PD1AB-6-K3 was further characterized in functional assays with whole blood from healthy donors and autoimmune disease patients (atopic dermatitis [AD], rheumatoid arthritis [RA], psoriasis [PS], Crohn's disease [CD], psoriatic arthritis [PsA], and systemic lupus erythmatous [SLE]). Whole blood was obtained from healthy donors and pre-screened autoimmune disease patients and heparinized (Sanguine Biosciences, Sherman Oaks, Calif.). The heparinized blood was diluted with RPMI-1640 medium, 200 μL was plated onto Covalink™ culture plates pre-coated with anti-CD3 antibody, and the plates dose titrated (100 nM to 0.1 nM) with the antibodies (PD1AB-6-K3, CTLA-4Ig (positive control), or hIgG1 Fc (negative control)). After 48 hours at 37° C., plasma was removed and assessed for IFN-γ and/or IL-17 release. The calculated EC₅₀ for IFN-γ or IL-17 was plotted per donor for a given autoimmune disease.

Overall, across normal and disease whole blood, immobilized PD1AB-6-K3 demonstrated activity in blocking T cell specific IFN-γ (FIG. 17A) and 1-17 (FIG. 17B) release. In normal donor whole blood, the average EC₅₀ for IFN-γ inhibition by PD1AB-6-K3 (n=4) was calculated to be 6.18±10.9 nM, whereas CTLA4-Ig, the positive control in this assay, had an EC₅₀ of 22.5±39.2 nM (data not shown). Coated hIgG1 Fc (negative control) exhibited no inhibitory activity (data not shown).

5.4.3 Effects on Cell Surface Expression of T Cell Inhibitory Receptors

T cell inhibitory receptors are co-inhibitory molecules that negatively interfere with T cell activation and function. The effect of PD1AB-6-K3 on expression of immune checkpoint proteins on T cells was evaluated using flow cytometry to measure cell surface expression of LAG-3, CTLA-4, and PD-1 under unstimulated and stimulated culture conditions. Human PBMCs were isolated from fresh buffy-coated whole blood (Blood Center of New Jersey, NJ; New York Blood Center, NY; Biological Specialty Corp., Colmar, Pa.). PMBCs (2×10⁶ cells) were pre-treated with PD1AB-6-K3-, control human IgG1- or PD-L1Fc-conjugated beads (Dynabead™ M-450 Epoxy, Invitrogen) at 37° C. for 24 hours. Cells were then stimulated with either anti-CD3 (0.3 μg/mL, R&D system, MAB100 ARH1113091) or a Staphylococcal enterotoxin B (SEB) solution (2 μg/μL Millipore, 11100-45-1) for 2 days at 37° C. (primary stimulation). Under unstimulated conditions (i.e., PD1AB-6-K3 treatment alone), LAG-3 expression was significantly inhibited on CD4+ memory T cells and there was a trend towards concentration-dependent reduction in LAG-3 expression on CD8+ memory T cells, while PD-1 surface expression was significantly decreased in both CD4+ memory T cells and CD8+ memory T cells (Tables 15 and 16). Both anti-CD3 and SEB-stimulated cells showed a significant reduction in LAG-3 expression on CD4+ memory T cells (Table 15). PD1AB-6-K3 had no effect on CD8+ T cell expression of either LAG-3 or CTLA-4 under primary stimulus conditions (Table 16). Similar to in vivo studies discussed below, PD1AB-6-K3 consistently down-regulated PD-1 expression in stimulated CD4+ and CD8+ T cells (see Tables 15 and 16). Number in parentheses indicates the number of donors; “Tet.” is the group treated with tetanus; “Time” is the time treated with PD1AB-6-K3 in “No stim.” group or time post-stimulation in all other groups; “NE” is no effect; “n/s” is not significant; * p≦0.05, ** p≦0.01, *** p≦0.001, **** p≦0.0001 versus media control by one-way ANOVA followed by Dunnett's Multiple Comparisons post-test.

TABLE 15 PD1AB-6-K3 Effect on Inhibitory Receptor Expression on CD4+ Memory T cells on Primary Stimulation. % Change (N) (increase (+), decrease (−)) PD1AB-6-K3 (nM) Time 0.5 5 50 0.5 5 50 0.5 5 50 Group (day) LAG-3 CTLA-4 PD-1 No 3 n/s −22(5) −21(5) NE NE NE −45(5) −55(5) −46(5) stim. * * **** **** **** 8 NE NE NE NE NE NE −18(6) −27(6) n/s * ** Anti- 2 −23(5) −23(5) −20(5) +9(6) +10(6) +9(6) −71(6) −76(6) −68(6) CD3 ** ** ** ** ** *** **** **** **** mAb 7 NE NE NE NE NE NE −56(6) −58(6) −55(5) *** *** *** SEB 2 −23(5) −26(5) −23(5) NE NE NE −65(5) −71(5) −63(5) **** **** **** **** **** **** 7 NE NE NE NE NE NE −43(6) −46(6) −39(6) *** ** ** Tet. 2 NE NE NE NE NE NE −43(3) −52(3) −40(3) ** ** 7 NE NE NE NE NE NE n/s −26(5) −20(5) ** *

TABLE 16 PD1AB-6-K3 Effect on Inhibitory Receptor Expression on CD8+ Memory T cells on Primary Stimulation. % Change (N) (increase (+), decrease (−)) PD1AB-6-K3 (nM) Time 0.5 5 50 0.5 5 50 0.5 5 50 Group (day) LAG-3 CTLA-4 PD-1 No 3 n/s n/s n/s NE NE NE −45(5) −55(5) −45(5) stim. *** **** ** 8 NE NE NE NE NE NE NE −16(6) NE * Anti- 2 NE NE NE NE NE NE −59(6) −62(6) −50(6) CD3 **** **** **** mAb 7 n/s +27(5) n/s NE NE NE −30(6) −35(6) −33(6) *** *** *** *** SEB 2 n/s n/s n/s NE NE NE −48(5) −55(5) −38(5) **** **** **** 7 NE NE NE NE NE NE −25(6) −30(6) −21(6) ** ** * Tet. 2 NE NE NE NE NE NE −41(3) −57(3) n/s ** ** 7 NE NE NE NE 20(5) NE NE −17(5) NE ** *

To achieve a secondary stimulus, human PBMCs described above were pre-stimulated with anti-CD3 (3 μg/mL) and anti-CD28 (1 μg/mL) for 24-48 hours at 37° C., then treated with PD1AB-6-K3 or human IgG1 isotype control at 37° C. for 24 hours. The cells were then re-stimulated with anti-CD3 (3 μg/mL) and anti-CD28 (1 μg/mL) for 6-24 hours. LAG-3 and CTLA-4 expression was evaluated by flow cytometry (BD LSRFortessa™ flow cytometer, FACSDiva™ analysis software (BD Bioscience) and Flow Jo Analysis software). Under secondary stimulus conditions, LAG-3 expression was increased in both CD4+ and CD8+ memory T cells, and CTLA-4 was increased in CD4+ memory T cells (data not shown).

T cell exhaustion in CD8+ T cells is characterized by down-regulation of memory markers such as interleukin-7 receptor (IL-7R) as well as up-regulation of inhibitory receptors (Blackburn et al., 2009, Nat. Immunol. U.S. 10(1):29-37). In chronic inflammatory conditions, PD-1 activity is proposed to attenuate T cell inflammatory function by inducing an exhausted T cell state associated with, for instance, dowregulation of memory markers such as IL-7R in CD8+ T cells. To evaluate effect of PD1AB-6-K3 on CD8+ T cell exhaustion, IL-7Rα (CD127) expression on activated T cells was measured in CD8+ T cells. CD8+ T cells were activated with anti-CD3 (R&D system, MAB100 ARH1113091) and anti-CD28 (eBiosciences, 16-0289) antibodies in combination with co-stimulatory signal through CD2 for 6 days. PD1AB-6-K3 reduced unexhausted IL-7R^(hi) CD8+ T cells and increased exhausted IL-7R^(lo) CD8+ T cells in an in vitro assay (FIG. 18). In FIG. 18, IgG1 represents non-specific human IgG1 isotype control; IgGFc represents non-specific human IgG1 Fc fragment control; *p<0.05; **p<0.01; ***p<0.001 by t test versus no Ab control (N=8); dagger symbol represents p value compared to isotype controls by Students t-test; † p<0.05; †† p<0.01; ††† p<0.001. These results indicate that PD1AB-6-K3 treatment increased the proportion of exhausted CD8+ T cells as defined by LAG-3 or CTLA-4 expression up-regulation or IL-7R expression down-regulation.

5.4.4 Inhibition of T Cell Proliferation

To evaluate the effects of PD1AB-6-K3 on T cell proliferation in the absence of influences from other cells, isolated CD4+ and CD8+ T cells were incubated with PD1AB-6-K3 bound to beads in cis (stimulus and PD1AB-6-K3 on the same bead surface) or trans (stimulus and PD1AB-6-K3 on separate bead surfaces). The effects were measured by tritiated-thymidine incorporation assay.

CD4+ and CD8+ T cells were enriched from fresh buffy-coat (leukocyte enriched units, Blood Center of New Jersey, NJ, New York Blood Center, NY and Biological Specialty Corp. Colmar, Pa.) by Ficoll-Hypaque density gradient centrifugation following incubation with EasySep™ negative selection. In brief, 2×10⁸ cells/ml PBMCs were mixed with 4 ml of Robosep™ buffer and transferred to a 14-ml polystyrene round bottom tube. EasySep™ Human enrichment cocktail (200 μl) was added per tube, vortexed, and incubated at room temperature for 10 minutes. EasySep™ Magnetic particles were added (300 μl per tube), vortexed, and incubated at room temperature for 5 minutes. Robosep™ buffer (5 ml) was added to each tube and mixed well by pipetting up and down. Tubes were placed in the silver magnet and incubated at room temperature for 5 minutes. The magnet and tube were picked up and in one continuous motion inverted, pouring off the desired fraction into a 50 mL conical tube. Cells were spun at 1200 rpm for 5 minutes. Supernatant was poured off and 5 ml of fresh media added. After cells were counted, an aliquot was removed for FACS analysis and the remaining cells were used for culture. Cells were isolated to ˜95% purity as determined by flow cytometry. Purified cells were plated at 1×10⁵ cells/well in a sterile 96-well plate at 100 μl per well in the cell-appropriate medium.

PD1AB-6-K3 was covalently coupled to Dynabeads M-450 Epoxy beads according to the manufacturer's protocol (Invitrogen). Briefly, 10′ beads were coated with 5 μg of antibodies. For “cis” configuration, 10′ beads were coupled with a mix of 1 μg of anti-CD3 antibody and 2 μg of PD1AB-6-K3. For “trans” configuration 10⁷ beads were coupled with 1 μg of anti-CD3 antibody, or 2 μg of PD1AB-6-K3. Covalent coupling of the proteins to the beads was performed in 0.1 mol/L sodium phosphate buffer for 24 hours at room temperature with gentle tilting and rotation. Dynabeads were resuspended in 1 ml phosphate buffered saline (PBS) with 0.1% bovine serum albumin (BSA), pH 7.4 to obtain 4×10 beads/ml.

Isolated CD4+ or CD8+ T cells (10⁶ cells) were resuspended in 100 μL of culture medium per well in 96-well plates. 100 μL of PD1AB-6-K3-conjugated beads were added at a bead-to-cell ratio of 2:1 or 8:1. Cells were incubated at 37° C. in a humidified incubator at 5% CO₂. On Day 2 of treatment for T cells and 24 hours prior to harvesting, one microcurie of 3H thymidine (Amersham) was added to each well and cells were incubated again at 37° C. in a humidified incubator at 5% CO₂. After 24 hrs of incubation with of 3H-thymidine, cells were harvested onto UniFilter™ GF/C filter plates (Perkin Elmer) using a cell harvester (Tomtec) and the plates were allowed to dry overnight. Microscint™ 20 (Packard) (20 μL/well) was added and plates were analyzed using a TopCount™ NXT (Packard) Microplate Scintillation and Luminescence Counter. Each well was counted for one minute and the counts per minute (cpm) values were plotted using GraphPad™ Prism.

In CD4+ T cells: At 1:2 and 1:8 cell to bead ratio, there was a significant proliferation inhibition for anti-CD3/PD1AB-6-K3 treated cells, compared to anti-CD3 treated cells for trans (% inhibition of 57±12 and 55±9 respectively [p<0.0001, N=4], whereas Cis configuration did not show significant inhibition (FIG. 19A).

In CD8+ T cells: At 1:2 and 1:8 cell to bead ratio, there was a significant proliferation inhibition for anti-CD3/PD1AB-6-K3 treated cells, compared to anti-CD3 treated cells for trans (% inhibition of 42±17 and 37±14, respectively [p≦0.05, N=4]), whereas cis configuration did not show significant inhibition (FIG. 19B).

These results demonstrate that PD1AB-6-K3 inhibited proliferation of anti-CD3 treated CD4+ and CD8+ T cells in the absence of influences from other cells, and that the inhibitory effect of PD1AB-6-K3 on T cell proliferation only occurred when PD1AB-6-K3 and anti-CD3 were in trans configuration.

5.4.5 Increase of CD25+Foxp3+ Induced Regulatory T Cells

To assess effect of anti-PD-1 antibodies on regulatory T cell (Treg cell) generation, naïve CD4+ T cells isolated from PBMC were induced to differentiate into induced Treg cells. Naïve CD4 T cells were negatively selected from PBMC using a naïve CD4+ T cell isolation kit (Miltenyi Biotec, Germany) following the manufacturer's instructions. Naïve CD4+ T cells were cultured with 0 to 2.5 ng/mL TGF-β, 10 ng/mL IL-2, 0.25 μg/mL soluble anti-CD28, and 1 μg/mL immobilized anti-CD3. Soluble PD1AB-6-IgG1 and IgG1 Fc isotypes were added at 1 nM. After 5 days of incubation, cells were collected and stained for Foxp3.

Cells were stained with diluted surface staining antibody (CD4 PerCP, CD25 PE, PD-1 BV421) for 30 minutes at 4° C., and were washed with 300 L of FBS. Cells were permeabilized and fixed with 500 μL of 1× Foxp3 fix/perm buffer per tube with 60 minutes incubation at room temperature. Cells were washed twice with 500 μL of perm buffer. Cells were stained intracellularly for Foxp3 with 50 μL of diluted Foxp3 antibody for 1 hour at room temperature before being washed with 500 μL of perm buffer. Cells were washed once with 500 μL of FACS buffer and resuspended in 175 μL of FACS buffer. Cells were transferred to 96-well plate for sample acquisition on a BD FACSCanto™ flow cytometer.

As shown in FIG. 20, PD1AB-6-IgG1 increased CD25+Foxp3+ induced Treg cells over a range of TGF-β concentration.

5.4.6 Expression of Immune Checkpoint Proteins on T Cells in Normal Donors and Autoimmune Disease Patients

PD-1 cell surface expression was compared between naïve and stimulated cells from normal donors and autoimmune disease patients (AD, CD, PS, PsA, RA, and SLE). In healthy donors the majority of memory T cells expressed PD-1, while only a few naïve T cells expressed PD-1 (data not shown). Patient-derived T cells expressed similar levels of PD-1 compared to healthy donor PBMC before and after activation with 5 μg/mL Phytohemagglutinin (Sigma, 61764-1MG) when cultured with 10% heat inactivated FBS (data not shown).

The expression of PD-1 and other checkpoint inhibitor protein on the surface of T cells may be differentially modulated in disease states and, by extension, have an impact on PD1AB-6-K3 activity. Expression of PD-1 and other immune checkpoint proteins on T cells from healthy controls and autoimmune disease patients (AD, RA, and PS) were assessed in ex vivo cultures of PBMCs. PBMCs (5×10⁵) from healthy and autoimmune disease donors (Conversant Biologics) were cultured in RPMI (Invitrogen) containing 10% heat-inactivated human autologous serum (Conversant Biologics). The PBMCs were either left unstimulated (basal conditions) or stimulated at 37° C. for 24 hours with anti-CD3 (3 μg/mL R&D system, MAB100 ARHI113091) and anti-CD28 (1 μg/mL, eBiosciences, 16-0289) antibodies. Cells were stained with LIVE/DEAD® dye (LIVE/DEAD® Fixable Blue Dead Cell Stain, Molecular Probes™ L23105) and then fluorescence-conjugated anti-CD4+(APC-Cy7, BD Biosciences 557871), anti-CD8+(PE-Cy5, BD Biosciences 555367), anti-PD-1 (BV650, BD Biosciences 564324), anti-PD-L1 (BV421, 563738), anti-PD-L2 (APC, BD Biosciences 557926), anti-LAG-3 (FITC eBiosciences 11-2239), anti-CTLA-4 (CD152) (BV786, BD Biosciences 563931) and anti-TIM-3 (PE-Cy7, eBiosciences 25-3109) antibodies for detection by flow cytometry (LSRFortessa flow cytometer, FACSDiva analysis software, BD Biosciences; FlowJo™ Analysis software).

On resting CD4+ T cells, the baseline expression of PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, and TIM-3 was not significantly different among the disease patients and healthy controls (HC) (Table 17). In contrast to CD4+ T cells, resting CD8+ T cells from AD and PS patients showed significantly lower PD-1 and TIM-3 expression, while RA patients were not different compared to healthy controls (Table 17).

In activated CD4+ T cells, PD-1 expression was upregulated in HC and PS patients versus unstimulated cells. The increase in PD-1 expression in the CD4+ T cells of PS patients was significantly higher than that of stimulated HC. Induction of PD-L1 upon T cell stimulation exhibited a similar pattern to PD-1. PS patient CD4+ T cells also showed a higher level of up-regulation of LAG-3, CTLA-4, and TIM-3 expression compared to HC. Expression of PD-L2 was not substantially changed after T cell stimulation in all groups, while AD samples had variable induced PD-1 and TIM-3 expression that did not reach significance (Table 18).

In activated CD8+ T cells, PD-1 expression trended higher relative to unstimulated CD8+ T cells, which already express PD-1 in their resting state; however, the increased expression did not reach significance. All three disease groups, AD, RA, and PS, that had showed reduced levels of PD-1 relative to healthy controls at basal unstimulated levels (Table 17), displayed higher levels of PD-1 on stimulated CD8+ T cells, though expression remained at or below that of unstimulated HC (Table 18). PD-L1 was significantly induced in activated CD8+ T cells compared to HC, AD, and PS patients (PD-L1 did not attain significance in RA patients) (Table 18). PD-L2 expression was also induced post CD8+ T cell stimulation in HC, but not disease patients (AD, RA, or PS) (Table 18).

The results indicated that circulating CD4+ T cells in AD, RA, and PS can express PD-1, PD-L1, PD-L2, CTLA-4, and TIM-3 when activated, with PS CD4+ T cells primed to express high levels of PD-1 and TIM-3 relative to those of healthy control. But, depending on the disease, CD8+ T cells are blunted in both their basal and inducible capacity to express PD-1 and TIM-3 following activation, depending on the disease.

TABLE 17 Baseline Expression of Immune Checkpoint Proteins on Resting T Cells from Autoimmune Disease Patients Checkpoint Proteins Autoimmune Diseases on CD4+ and Atopic Dermatitis Rheumatoid Arthritis Psoriasis CD8+ T Cells Percent of Healthy Control Samples CD4+ T cells PD-1 102 ± 6 120 ± 11 112 ± 7  PD-L1  94 ± 8 109 ± 13 116 ± 16 PD-L2 116 ± 9 142 ± 19 157 ± 40 LAG-3 107 ± 9 117 ± 8  115 ± 8  CTLA-4  151 ± 62 115 ± 40  92 ± 27 TIM-3  67 ± 17 90 ± 7 100 ± 16 CD8+ T cells PD-1   51 ± 19*  75 ± 17  61 ± 9** PD-L1  111 ± 17 123 ± 24 156 ± 22 PD-L2 115 ± 9 111 ± 35 101 ± 18 LAG-3 117 ± 7 129 ± 14 121 ± 6  CTLA-4  119 ± 5** 103 ± 12 116 ± 4* TIM-3   32 ± 18**  78 ± 32  69 ± 9**

TABLE 18 Expression of Immune Checkpoint Proteins on Stimulated T Cells From Autoimmune Disease Patients Relative to Stimulated Healthy Controls Checkpoint Proteins Autoimmune Diseases on CD4+ and Atopic Dermatitis Rheumatoid Arthritis Psoriasis CD8+ T Cells Percent of Healthy Control Samples CD4+ T cells PD-1 154 ± 26 130 ± 25   227 ± 21** PD-L1 120 ± 27 83 ± 40  216 ± 21** PD-L2 116 ± 11 113 ± 4  111 ± 6  LAG-3 146 ± 28 109 ± 28  162 ± 13* CTLA-4 102 ± 20 74 ± 18  216 ± 36** TIM-3 96 ± 4 88 ± 11 153 ± 10* CD8+ T cells PD-1  72 ± 28 59 ± 8  64 ± 8* PD-L1  229 ± 58* 150 ± 89  267 ± 46* PD-L2 123 ± 34 76 ± 22  56 ± 10* LAG-3  97 ± 21 87 ± 22 144 ± 16  CTLA-4  99 ± 25 124 ± 13  138 ± 7*  TIM-3  85 ± 35  46 ± 12* 52 ± 6*

5.4.7 Expression of PD-1 and PD-L1 in Rheumatoid Arthritis, Crohn's Disease, and Psoriasis Disease Tissues

Expression of PD-1 and PD-L1 was investigated in a small subset of rheumatoid arthritis patients (n=5), Crohn's disease patients (n=3), and Psoriasis patients (n=3), as compared to their normal counterparts (n=3) using immunohistochemistry.

Formation of well-organized lymphoid follicles at the site of disease is common in Crohn's disease and rheumatoid arthritis but not in psoriasis, suggesting that PD-1 and PD-L1 expression, response, and dynamic during disease progression may differ among these chronic indications. PD-1 and PD-L1 expression scores (FIGS. 25A-25C) show marked upregulation of PD-1 (2-fold increase) and PD-L1 (6-fold increase) in Crohn's (FIG. 25A), and subtle changes in Psoriasis (FIG. 25C).

In general, PD-1 is expressed in lymphocytes present within organized lymphoid follicles but not in lymphocytes in diffuse infiltrates (FIGS. 21-23, left panels). PD-L1 is, in turn, more widely expressed and is found in leukocytes within the lymphoid follicles (FIG. 22, right panel) and also diffusely distributed in diseased tissues (FIG. 23, right panel). PD-L1 is also expressed by other cell types such as stromal fibroblasts and endothelial cells in areas of intense inflammation. FIG. 24, right panel shows details of marked upregulation of PD-L1 expression in area of intestinal ulceration in a Crohn's patient. The large cells have morphology compatible with that of activated macrophages.

FIGS. 21 and 22 show representative IHC staining in two RA patients. As shown in FIG. 21, while PD-1 is strongly expressed in the cytoplasm of lymphocytes within a lymphoid aggregate in the synovium of an RA patient (left panel), expression of PD-L1 is weak and restricted to a few large cells (likely macrophages/dendritic cells) within the lymphoid aggregates (right panel). As shown in FIG. 22, a few lymphocytes express cytoplasmic/membrane PD-1 within a lymphoid aggregate of an RA patient (left panel). PD-L1 is weakly expressed in the cytoplasm and membrane of macrophages and/or dendritic cells in a similar location.

FIG. 23 shows representative IHC staining in the intestines of Crohn's patient. As shown in FIG. 23, expression of PD-1 (left panel) is restricted to lymphocytes within follicular aggregates in the mucosa (top left) and submucosa (bottom center) in Crohn's disease patient. PD-L1 (right panel), on the other hand, is more widely expressed by macrophages/dendritic cells in lymphoid aggregates and in diffuse mucosal inflammatory infiltrates.

FIG. 24 shows representative IHC staining in ulcerated region of Crohn's disease intestinal sample. As shown in FIG. 24, in areas of ulceration where the inflammatory infiltrate is diffuse without follicular formation, there are rare PD-1+ lymphocytes (left panel) but abundant PD-L1+ macrophages (right panel). Expression of PD-1 is predominantly cytoplasmic whereas PD-L1, when upregulated, appears predominantly membranous.

FIGS. 25A-25C show PD-1 and PD-L1 expression scores in Crohn's, RA, and psoriasis patients. Significant upregulation of PD-L1 was observed in Crohn's (FIG. 25A), but subtle increases in PD-L1 expression was observed in psoriasis (FIG. 25C).

5.4.8 Inhibition of Differentiation of B Cells into Plasmablasts

CD19+ B cells were purified from normal human PBMCs using EasySep™ Human B Cell Enrichment Kit from StemCell Technologies (Vancouver, Canada). CD3+ T cells were purified from normal human whole blood using EasySep™ Direct Human T Cell Enrichment Kit from StemCell Technologies.

Human anti-CD3 antibody UCHT1 (BioLegend, San Diego, Calif.), PD1AB-6-K3, or PD-L1 Fc fusion protein was covalently coupled to Dynabead M-450 Epoxy beads according to the manufacturer's protocol (Invitrogen, Carlsbad, Calif.). For “trans” configuration coupled beads, 10⁷ beads were coupled with either 1 μg of anti-CD3 antibody, or 2 μg of PD1AB-6-K3 or PD-L1 Fc fusion protein such that each protein represents a distinct bead set with only one protein type being conjugated on a single bead. Covalent coupling of the proteins to the beads was performed in 0.1 mol/L sodium phosphate buffer for 24 hours at room temperature with gentle tilting and rotation. Protein-coupled Dynabeads were centrifuged and resuspended in 1 mL blocking buffer (phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), pH 7.4) to obtain 4×10 beads/mL. Null beads were made by incubating the beads in the blocking buffer only.

Enriched normal human CD19+ B and CD3+ T cells were cultured in the in vitro B cell differentiation co-culture system. CD19+ B cells were added to each well of a 96-well round-bottom plate (final cell count=2×10⁵/well). CD3+ T cells were added to each well of a 96-well round-bottom plate (final cell count=1×10⁵/well). Bead-bound PD1AB-6-K3, PD-L1 Fc fusion protein, or null beads was added to cells at a bead to cell ratio of 4 to 1.

After 1 hour, bead-bound human anti-CD3 antibody UCHT1 (bead to cell ratio of 2 to 1) and recombinant human IL-21 (100 ng/mL, ThermoFisher Scientific, Waltham, Mass.) were added to each well and incubated at 37° C. for 4 days. On day 4, additional recombinant human IL-21 (100 ng/mL) was added to each well and incubated at 37° C. for 2 days.

Cells were harvested at day 6. Then the cells were stained with LIVE/DEAD Fixable blue dead cell stain (ThermoFisher Scientific). Next, the cells were blocked with BD Pharmigen™ Fc block (BD Bioscience, San Diego, Calif.). Following blocking, BD Bioscience flow antibodies (CD20-APC, CD38-BV510, IgM-BUV395, CD3-PERCP5.5, CD27-BV786) were added according to experimental design for surface marker staining. The samples were immediately analyzed or put at 4° C. overnight. All samples were analyzed using a BD LSRFortessa™ cell analyzer, FACSDiva™ analysis software (BD Bioscience), and FlowJo® Analysis software (FlowJo LLC, Ashland, Oreg.). Plasmablasts were defined as CD20-CD38+ B cells within the total CD19+ B-cell population.

Supernatants were harvested at Day 6 and analyzed by human IgG ELISA kit (Abcam, Cambridge, UK) for IgG production.

FIG. 33 shows exemplary flow cytometry results from four donors that demonstrate differentiation of CD20+CD38-activated B-lymphocytes into CD20-CD38+ plasmablasts in the in vitro T cell/B cell co-culture system.

In the T cell receptor activated CD3+ T Cell/total B-Cell co-cultures, bead-bound PD1AB-6-K3 induced average 25.0% reduction (p=0.15) of plasmablast numbers (FIG. 34A), average 24.4% reduction (p=0.04) of IgG secretion (FIG. 34B), but average 11.5% increase (p=0.15) of IgM surface expression (FIG. 34C), compared to null beads. The percentage change in each donor sample is listed in Table 22 below.

TABLE 22 PD1AB-6-K3-Induced Percentage Change of Plasmablast Numbers, IgG Secretion, and IgM Surface Expression in T Cell Receptor Activated CD3+ T Cell/Total B-Cell Co-cultures % Change of % Change of % Change of IgM Donor Plasmablast Numbers IgG Secretion Surface Expression 1 −2.3 9.5 −0.9 2 19.3 −4.6 2.4 3 −77.8 −95.7 20.9 4 −67.5 −2.8 −1.2 5 −51.1 −19.9 −13.2 6 42.2 −16.2 27.9 7 −0.1 −34.0 32.7 8 −62.5 −31.3 23.4

In the parallel T cell receptor activated CD3+ T Cell/total B-Cell co-cultures, bead-bound PD-L1 Fc fusion protein induced average 43.8% reduction (p=0.02) of plasmablast numbers (FIG. 35A), average 12.7% reduction (p=0.38) of IgG secretion (FIG. 35B), but average 0% increase (median 6.7% increase, p=0.55) of IgM surface expression (FIG. 35C), compared to null beads. The percentage change in each donor sample is listed in Table 23 below.

TABLE 23 PD-L1 Fc Fusion Protein-Induced Percentage Change of Plasmablast Numbers, IgG Secretion, and IgM Surface Expression in T Cell Receptor Activated CD3+ T Cell/Total B-Cell Co-cultures % Change of % Change of % Change of IgM Donor Plasmablast Numbers IgG Secretion Surface Expression 1 −18.9 −3.6 −0.5 2 −13.2 18.4 9.1 3 −93.4 −69.2 4.4 4 −98.8 −25.5 −58.2 5 −72.3 −17.1 −17.5 6 −2.8 37.2 17.8 7 12.7 −15.6 21.6 8 −63.5 −25.6 23.4

These results demonstrate that PD1AB-6-K3 inhibits differentiation of B cells into plasmablasts and secretion of IgG in the in vitro CD3+ T cells/total B cells co-culture system, suggesting that PD1AB-6-K3 may be effective in treating B cell antibody-mediated diseases, such as allergy.

5.5 In Vivo Pharmacology

5.5.1 Xeno-Graft Versus Host Disease (GvHD) Models

Xeno-GvHD model involves transfer of human PBMC or human T cell subsets (isolated by purification or depletion of specific T cell subsets) into immunodeficient NSG strain of mice. Transfer of human PBMC or T cells into immunodeficient NSG mice leads to engraftment of human T cells into lymphoid organs, activation of human T cells measurable as cytokine secretion, and infiltration of human lymphocytes into various organs. In efficacy studies, T cell cytokine levels in plasma (IFN-γ, GM-CSF, 11-17, and TNF-α) and human cell engraftment (measured as % human CD45+) in blood and/or spleen, were used as primary endpoints. In some studies, additional characterization of lymphocytic infiltration into non-lymphoid organs was performed to better understand the biology of the test molecules. This model was chosen due to lack of cross-reactivity of PD1AB-6-K3 to mouse PD-1. In all of these studies, CTLA4Ig was used as a positive control for the model.

5.5.1.1 Effect of PD1AB-6-K3 in Human PBMC Transfer Models of T Cell Activation

Studies were conducted to determine the in vivo efficacy of PD1AB-6-K3 on human T cell activation. Human PBMCs were transferred into NSG knockout mice and T cell activation determined by cytokine release into the plasma and human CD45+ lymphocyte engraftment in the blood and spleen of the mice. Twenty million human PBMCs (0.1 mL) from healthy donors (AllCells Inc., Alameda, Calif.) were administered intravenously by injection into the tail of NSG mice (Jackson Laboratories, Bar Harbor, Me.) on Day 0. On Day 1, the appropriate test article (PD1AB-6-K3, PD1AB-6-IgG1, PD1AB-6-4P, CTLA4Ig/abatacept positive control (Bristol-Myers Squibb) and human IgG1 isotype negative control (Eureka Therapeutics)) was administered at 2.5 mg/kg, 3 times a week (Monday, Wednesday, and Friday), intraperitoneally (IP) for 28 days in Studies I and II and for 16 days in Study III. After 28 days, the blood and spleen of the mice were collected for preparation of plasma and cell suspensions. The levels of human cytokines GM-CSF, IFN-γ, TNF-α, IL-17 were measured in mouse plasma via a custom human Meso Scale™ 4 spot enzyme-linked immunosorbent assay (ELISA) kit (Meso Scale Diagnostics, Rockville, Md.) using a Meso Scale™ Sector Imager 6000. Plasma levels of the cytokines were determined according to a standard curve (5.000 to 0.3 pg/mL). Efficacy was expressed as percent inhibition compared to vehicle control. Engraftment of human CD45+ cells was assessed in the xeno-GvHD mouse model by determining the percentage increase in human CD45+ cells by flow cytometry. One hundred microliters of each spleen cell suspension or whole blood sample was stained with anti human CD45-PerCP for flow cytometry analysis (BD FACSCalibur™, FlowJo™ software, BD Biosciences).

The studies demonstrated that the level of human lymphocyte engraftment (human CD45+ cells) and the levels of human cytokines such as GM-CSF, IFN-γ, TNF-α, IL-10, and IL-17 detected in mouse plasma varied with the donor. PD1AB-6-K3 had minimal effect on human CD45+ lymphocyte engraftment whereas CTLA4Ig significantly reduced the extent of engraftment (FIG. 26B). PD1AB-6-K3 showed a trend towards inhibition of cytokines in one study and statistically significant inhibition of cytokines (GM-CSF (FIG. 26A), IFN-γ, TNF a, IL-10, and IL 17 [p<0.0001 to 0.05]) in another study with a different donor. Contrary to expectation, the study with higher cytokine levels showed more inhibition with PD1AB-6-K3. PD1AB-6-K3 treatment significantly reduced PD-1 expression on both CD4+ and CD8+ T cells in blood and spleen in both studies (data not shown). The differential gene expression explored in T cells isolated from spleens did not show any statistically significant differences between PD1AB-6-K3 (5 mg/kg) versus vehicle-treated CD4+ or CD8+ T cells. However, analysis of specific genes of interest related to the hypothesized drug action and a pathway analysis approach revealed a number of pathways and functional gene groups significantly altered by PD1AB-6-K3 treatment (data not shown). Table 19 shows representative data from Study I and Study II.

TABLE 19 Summary of results testing PD1AB-6 variants in Human PBMC transfer model Vehicle data - cytokine (pg/ml); Treatment data - % inhibition vs Vehicle Human cell engraftment * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 Dosing Dose/mouse Human Human Human Human Spleen - Spleen - frequency Treatment (~mpk) IFN TNF GM-CSF IL-17 % hu CD45 % hu CD4 Study I Vehicle 1228 <10    397.3   25.2 64% 24% M, W, F PD1AB-6-IgG1 50 ug  54%, % inhibition 53%  25% 52% 27% (~2.5 mpk) * not PD1AB-6-K3 50 ug 25% calculated 27%  58% 59% 24% (~2.5 mpk) when PD1AB-6-4P 50 ug 50% cytokine 47%  50% 49% 25% (~2.5 mpk) levels <10 pg/ml CTLA4Ig 50 ug 100%,  100%,   98%, 7% 21% (~2.5 mpk) **** *** ** Study II Vehicle 3288   28.7 1787 152 72% 52% M, W, F Human IgG1 50 ug No 28 No No 69% 50% Isotype (~2.5 mpk) inhibition inhibition inhibition PD1AB-6-IgG1 50 ug 30% 44%  54%,  57%, 63%  40%, (~2.5 mpk) ** * * PD1AB-6-K3 50 ug 41% 64%, 66%,  62%,   62.00%  41%, (~2.5 mpk) ** *** * * PD1AB-6-4P 50 ug 11% 50%, 52%, 41%  56%, 47% (~2.5 mpk) * ** * CTLA4Ig 50 ug 100%,  99%, 100%,   81%,  13%, 44% (~2.5 mpk) *** **** **** *** ****

PD1AB-6-K3 inhibited T cell cytokine production, which was comparable to inhibition of cytokine levels seen with the other two variants PD1AB-6-IgG1 and PD1AB-6-4P, indicating that PD1AB-6 activity was not due to complement mediated depletion of T-effector cells (Table 19). In addition, there was no effect on the percentage of human CD45+ cells engrafted with any of the PD1AB-6 antibodies tested, further supporting the hypothesis that Clq (complement) dependent cell depletion was not occurring (small but statistically significant inhibition of CD45+ cell engraftment with PD1AB-6-4P seen in this study is not seen consistently across studies). The positive control CTLA4Ig at a comparable dose prevented the engraftment of human cells, leading to no detectable cytokine production.

5.5.1.2 Effect of PD1AB-6-K3 in Human CD4+ T Cell Transfer Models of T Cell Activation

To specifically test the effect of PD1AB-6-K3 on CD4+ T cells, which are the primary mediators in a number of autoimmune diseases, studies were done by transferring purified human CD4+ T cells into NSG knockout mice to determine plasma cytokine levels and the percentage of human CD45+ cells in the blood and spleen. CD4+ cells were purified from human PBMCs (AllCells) using mycobacterium avium complex (MACS) magnetic separation systems (Miltenyi, Auburn, Calif.). Five million purified human CD4+ T cells were injected intravenously (IV) into NSG female mice on day 0 and, one day later, PD1AB-6-K3 was dosed at 2.5 mg/kg, IP, 3 times a week. After 28 days, the blood and spleen of the mice were collected for preparation of plasma and cell suspensions. Analysis of plasma cytokines levels and CD45+ cell engraftment was done as discussed previously (see section 5.5.1.1).

PD1AB-6-K3 significantly inhibited human CD45+ lymphocyte engraftment (>75%) compared to vehicle control or human IgG1 isotype control. This inhibition of CD45+ cell engraftment was not seen in PBMC transfer model, indicating that signals from non-CD4+ T cells had an impact on PD1AB-6-K3-mediated inhibition of human CD45+ lymphocyte engraftment. Compared to the vehicle control group, PD1AB-6-K3 at 2.5 mg/kg given 3 times a week inhibited human GM-CSF (98%), IFN γ (99%), and 11-17 (98%) production (p<0.0001). With a C_(max) at 4 hours following a single IP dose of 2.5 mg/kg, the levels of PD1AB-6-K3 were 5 μg/mL at 72 hours post-dose and were detectable for up to 168 hours. Table 20 shows representative data from these studies.

TABLE 20 Summary of results testing PD1AB-6 variants in Human CD4+ T cell transfer model Vehicle data - cytokine (pg/ml); Treatment data - % inhibition vs Vehicle Human cell engraftment * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 Human Spleen - Dosing Dose/mouse Human GM- Human % hu Spleen - % hu frequency Treatment (~mpk) Human IFN TNF CSF IL-17 CD45 CD4 Study IV Vehicle  1364 <10   355   146.7 45%  49% M, W, F Human IgG1 50 ug (~2.5 mpk) 28%  % 17%  32%  35%  48% Isotype inhibition PD1AB-6-IgG1 50 ug (~2.5 mpk) 99%, not 98%, 99%, 20%, 41% **** calculated **** **** *** PD1AB-6-K3 50 ug (~2.5 mpk) 99%, when 98%, 99%, 11%, 50% **** cytokine **** * **** PD1AB-6-4P 50 ug (~2.5 mpk) 95%, levels 93%, 98%, 24%, 43% **** <10 pg/ml **** *** ** CTLA4Ig 50 ug (~2.5 mpk) 100%,  100%,  100%,   1%, 59% **** **** **** **** Study V Vehicle 95431 51.2 961.9 146.4 39%  Not Calculated M, W, F (much higher value with V-plex MSD) PD1AB-6-K3 25 ug (~1.25 mpk) Not Calculated 86%  89%, 98%, 11%, Not Calculated **** **** **** PD1AB-6-K3 12.5 ug (~0.6 mpk) Not Calculated 93%, 95%, 98%, 11%, Not Calculated **** **** **** **** PD1AB-6-4P 25 ug (~1.25 mpk) Not Calculated 85%, 87%, 93%, 12%, Not Calculated **** **** **** **** PD1AB-6-4P 12.5 ug (~0.6 mpk) Not Calculated 87%, 88%, 97%,  9%, Not Calculated **** **** **** **** CTLA4Ig 25 ug (~1.25 mpk) Not Calculated 99%, 99%, 99%,  3%, Not Calculated **** **** **** **** CTLA4Ig 12.5 ug (~0.6 mpk) Not Calculated 98%, 98%, 97%,  5%, Not Calculated **** **** **** ****

FIGS. 27A-27B and 28A-28C show representative data from Study V. PD1AB-6-K3 at a dose level as low as 0.63 mg/kg, IP, given 3 times a week, significantly inhibited CD45+ engraftment in both blood (84%, p<0.0001, FIG. 27A) and spleen (73%, p<0.0001, FIG. 27B), and plasma levels of GM-CSF, TNF-α, and 1-17 compared to vehicle treatment (86% to 98% reduction, p<0.0001) (FIGS. 28A-28C). This indicated that the minimal efficacious dose of PD1AB-6-K3 in the CD4+ T cell transfer model will likely be <0.63 mg/kg, 3 times a week.

5.5.1.3 Effect of PD1AB-6-K3 in CD8+ T Cell-Depleted Human PBMC Transfer Models of T Cell Activation

Studies were also conducted in modified xeno-GvHD model established by the transfer of CD8+ T cell-depleted PBMCs which allowed an assessment of the effects of PD1AB-6-K3 on CD4+ T cells in the presence of co-stimulatory and survival signals from non-T cells. CD8+ cells were magnetically labeled in the fresh human PBMCs (AllCells), using the MACS CD8 Microbeads in the BSL2 laboratory. Once labeled, the cell suspension was loaded onto a MACS separation column on a magnetic field, which retained the CD8+ cells, or purified using a QuaroMACS™ Separator (Miltenyi). Unlabeled cells that were CD8 negative were collected and pooled. At the start (Day 0) of the experiment, 0.1 mL of cell suspension (20 million CD8+ T cell-depleted PBMCs per animal) was injected IV into the tail vein of NSG female mice. In Study VI, VII, VIII preventive dosing, and IX preventive dosing, administration of PD1AB-6-K3, PD1AB-6-4P, CTLA4Ig, or PD-L1 Fc started on Day 1 and the study ended on Day 16. In Study VIII therapeutic dosing, administration of PD1AB-6-K3, PD1AB-6-4P, CTLA4Ig, or PD-L1 Fc started on Day 15 and the study ended on Day 28. Analysis of plasma cytokines levels and CD45+ cell engraftment was done as discussed previously (see section 5.5.1.1).

A starting dose level of 0.63 mg/kg, given 3 times a week, was chosen based on the lowest dose tested in the CD4+ T cell transfer model. There was no inhibition of human CD45+ cell engraftment in either blood or spleen at Day 16 (study termination) with PD1AB-6-K3 treatment. In Study VI, both doses of PD1AB-6-K3 (0.63 mg/kg and 0.3 mg/kg) showed significant inhibition of GM-CSF (79% at 0.63 mg/kg; 71% at 0.3 mg/kg; p<0.0001), IFN-γ (60% at 0.63 mg/kg; 51% at 0.3 mg/kg; p<0.05), and IL-17 (87% at 0.63 mg/kg; 66% at 0.3 mg/kg; p<0.01 to 0.05). In another study VII with a different donor, lower doses of 0.3 and 0.15 mg/kg given 3 times a week were tested (data not shown). There was inhibition of some cytokines (GM-CSF and 1-17 at both doses, TNF-α with 0.15 mg/kg only), but no inhibition of IFN-γ at any dose. However, treatment with CTLA4-Ig inhibited T cell engraftment and suppressed cytokine release at a dose as low as 0.15 mg/kg. In both studies VI and VII, PD1AB-6-K3 significantly down-regulated PD-1 expression on CD4+ T cells at all doses tested.

PD1AB-6-K3 dosed at 0.63 mg/kg weekly, showed inhibition of GM-CSF (p<0.05) and IL-17 (p<0.01) (Study VIII preventive dosing) but only IL-17 inhibition (p<0.05) with a different donor PBMC (Study IX preventive dosing). Similar to the results with 3 times a week dosing, once weekly treatment with PD1AB-6-K3 had no impact on engraftment of human CD45+ cells. In Study VIII therapeutic dosing, dosing at 1.25 mg/kg 3 times a week with PD1AB-6-K3 or CTLA4-Ig post-disease onset (15 days after cell transfer, considered therapeutic dosing), only inhibited GM-CSF and was considered not efficacious in therapeutic dosing in the CD8+ T cell depleted human PBMC transfer model. There was a significant reduction in human PD-1 expression by 46% on CD4+ T cells in spleen with PD1AB-6-K3 treatment (p<0.05, data not shown). Based on the cytokine inhibition results of all of the above studies, PD1AB-6-K3 at 0.63 mg/kg, IP, 3 times a week, was identified as the minimal efficacious dose in the CD8+ T cell-depleted human PBMC transfer model. Tables 21A and 21B show representative data from Studies VII-IX.

TABLE 21A Summary of results testing PD1AB-6 variants in Human CD8+ T cell-depleted PBMC transfer model Vehicle data - Human cell cytokine level in pg/ml; engraftment Treatment data - (as % of total % inhibition vs Vehicle lymphoctyes) * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 Dosing Dose/mouse Human Human Human Human Spleen - Spleen - frequency Treatment (~mpk) IFN TNF GM-CSF IL-17 % hu CD45 % hu CD4 Study VI Vehicle 5452 <10 124.3 28 53% 37% M, W, F PD-L1 Fc Protein 50 ug % 30%   3% 57% 39% (~2.5 mpk) inhibition PD-L1 Fc Protein 25 ug 17%  not 28%  61% 42% (~1.25 mpk) calculated PD-L1 Fc Protein 12.5 ug when 17%   4% 62% 42% (~0.6 mpk) cytokine PD1AB-6-K3 12.5 ug 60%, levels 79%, 87%, 61% 35% (~0.6 mpk) * <10 pg/ml **** ** PD1AB-6-K3 6.25 ug 51%, 71%, 66%, 59% 40% (~0.3 mpk) * **** * CTLA4Ig 12.5 ug 99%, 99%, 98%, 31% 64% (~0.6 mpk) *** **** ** Study VIII Vehicle  464 <10 290.5 74.9 pg/ml 58% 48% Q7D, PD1AB-6-K3 12.5 ug 32%  % 43%, 55%, 59% 47% preventive (~0.6 mpk) inhibition * ** dosing PD1AB-6-4P 12.5 ug not 41%, 57% 47% (~0.6 mpk) calculated * CTLA4Ig 12.5 ug 79%, 83%, 87%, 45% 41% (~0.6 mpk) ** **** ****

TABLE 21B Summary of results testing PD1AB-6 variants in Human CD8+ T cell-depleted PBMC transfer model Human cell Vehicle data - engraftment cytokine level in pg/ml; (as % of Treatment data - total % inhibition vs Vehicle lymphoctyes) * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 Study VIII Vehicle 25 ug 4418  44 1776 233  70% 67% M, W, F; (~1.25 mpk) therapeutic PD1AB-6-K3 25 ug 2% 17% 24%, 18%  76% 74% dosing (~1.25 mpk) * PD1AB-6-4P 25 ug 0%  0%  2% 0 66% 69% (~1.25 mpk) CTLA4Ig 25 ug 6% 24% 35%, 0 73% 68% (~1.25 mpk) ** Study IX Vehicle  409 <10  332 15.7 pg/ml 54% 82% Q7D, PD1AB-6-K3 12.5 ug 3% % inhibition 28%  61%, 56% 80% preventive (~0.6 mpk) not * dosing PD1AB-6-4P 12.5 ug calculated 30%  57% 79% (~0.6 mpk) CTLA4Ig 12.5 ug 84%,  89%, 85%, 41% 82% (~0.6 mpk) * * **

As shown above, PD1AB-6-K3 treatment reduced the overall engraftment efficiency when purified CD4+ T cells were transferred into NSG mice, but did not impact the engraftment of human CD45+ lymphoid cells or alter the CD4:CD8 percentages within the engrafted cells when PBMC or CD8+ T cell depleted PBMC were engrafted (Tables 19-21). This indicated that the interactions with receptors on other cell types (primarily monocytes in CD8+ T cell-depleted PBMC) influence the inhibitory signals from PD1AB-6-K3.

5.5.1.4 Downregulation of PD-1 Expression Precedes Cytokine Inhibition

Five million purified human CD4+ T cells were injected IV into NSG female mice on day 0. The mice were given a single dose of 50 μg/mouse of TAPA antibodies on day 21 posT cell transfer. PK samples were collected up to day 7. Cytokine levels and PD-1 expression were measured 24 h after the last dose. Cells were stained with anti-human CD45 and anti-human CD4 (to distinguish human CD4+ T cells) and anti-PD-1 (eBioscience antibody shown to bind PD-1 in the presence of PD1AB-6 variants) to measure PD-1 expression. Graph shows mean fluorescence intensity staining of PD-1 on CD4+ T cell surface. As shown in FIGS. 29A-29B, the PD-1 downregulation was seen as early as 24 h after a single dose of PD-1 antibody (FIG. 29A), and seen before cytokine inhibition could be detected (FIG. 29B).

5.5.1.5 Correlation of Efficacy (Cytokine Inhibition) with PD-1 Downregulation

Correlation between PD-1 downregulation and cytokine inhibition (efficacy) was plotted across various studies (FIGS. 30A-30B). PD1AB-6-K3 showed a trend towards more enhanced down-regulation of PD-1 expression as well as more cytokine inhibition as compared to PD1AB-6-4P. This was especially true for IL-17 inhibition (FIG. 30A). PD-L1.Fc, which showed no inhibition of cytokines, also did not downregulate PD-1 (FIGS. 30A-30B).

5.5.2 Experimental Autoimmune Encephalomyelitis (EAE) Model in Human PD-1 Knock-in (hPD-1 KI) Mice

Using a murine surrogate of PD1AB-6-K3, the in vivo efficacy of PD1AB-6-K3 was further tested in mouse models of EAE. A human PD-1 knock-in homozygous (hPD-1 KI) mouse on C57BL/6 background was used for these studies. PD1AB-6-K3 was modified to retain the PD-1 binding variable region, with Fc region switched to murine IgG2a with mutations comparable to K322A. This molecule is referred as PD1AB-6-K3 muFc and served as a surrogate molecule for testing PD1AB-6-K3 activities in hPD-1 KI mice. Parental PD1AB-6-IgG1 and PD1AB-6-K3 muFc were assessed for efficacy in two versions of the mouse model of EAE induced by myelin oligodentrocyte glycoprotein (MOG) peptide, with either pertussis toxin (PTX) or anti-CD25 administration in female hPD-1 KI mice (MOG-EAE).

For the PTX administration version, mice were anesthetized with inhaled isoflurane and given two subcutaneous (SC) injections of 100 μL each of MOG peptide 35-55 (MOG) emulsed in complete Freund's adjuvant (CFA) from Hooke Laboratories (Lawrence, Mass.) for a total of 200 μL MOG/CFA injection on Day 0. One injection was given on the upper, and the second on the lower, back area (100 μL per injection site). Two hours post-immunization, the first intraperitoneally (IP) administration of PTX (200 ng in 0.1 mL) (Hooke Laboratories) was given to each animal. The second IP administration of PTX (200 ng in 0.1 mL) was given 24 hours later.

For the anti-CD25 administration version, mice were anesthetized with inhaled isoflurane and given one SC injection of 100 μL MOG/CFA on Day 0 on the lower back of the mouse. On Day 6, each mouse was injected with 1 mg of anti-mouse CD25 antibody (Bio X Cell, West Lebanon, N.H.) IP in a volume of 200 μL. The second injection of 100 μL MOG/CFA was given on Day 7 in the upper back area.

Parental PD1AB-6-IgG1 was administered IP on Monday, Wednesday, and Friday at a dose of 10 mg/kg, starting at Day 5 post-initiation of the study. PD1AB-6-K3 muFc was administered IP on Monday, Wednesday, and Friday at a dose of 1.25 mg/kg, 5 mg/kg, or 10 mg/kg, starting Day 5 (prophylactic treatment) or Day 12 (therapeutic treatment) post-initiation of the study.

In the EAE model, where mice were induced to develop chronic EAE after injection with MOG/CFA followed by 2 injections of PTX, there was significant mortality in treatment groups dosed with PD1AB-6-K3-IgG1 or PD1AB-6-K3 muFc compared to vehicle group. The mortality was not associated with higher disease score and occurred within a few hours of antibody injection.

To determine whether the deaths in the MOG-EAE model with PTX were target-related, additional studies were designed based on the literature (Ji et al., 2011, J Immunol 186(5):2750-56). A rat anti-mouse integrin alpha (very late antigen [VLA])-4 antibody (EMD Millipore, Billerica, Mass.) and a corresponding rat isotype control antibody were used in the MOG-EAE model with PTX. In line with the literature, treatment with anti-VLA-4 and rat isotype control antibody from Day 5 led to significant mortality compared to vehicle control, with death occurring within a few hours of antibody injections, and with a lack of correlation between mortality and disease score. These observations were similar to those made with PD1AB-6-K3 muFc, suggesting mortality was not target-related. The MOG-EAE model was modified to replace PTX with depleting anti-CD25 as an adjuvant based on the literature (Montero et al., 2004, J Autoimmun. 23(1):1-7).

In the modified anti-CD25 EAE model, where mice were induced to develop chronic EAE after injection with MOG/CFA and anti-CD25, clinical EAE (as assessed by body weight change and disease score) was evident by Day 11 to Day 16 and peaked around Day 20 in the vehicle group. Two animals from vehicle saline group were euthanized on Day 24 and Day 27, due to excessive wounds in the feet and tail region. One animal each from isotype control (mouse IgG2a, Bio X Cell) 5 mg/kg group and PD1AB-6-K3 muFc 5 mg/kg group were found dead on Day 33. Both these mice had a high disease score of 4, and the death was likely related to EAE disease. Since mortality was seen both in mouse IgG2a and PD1AB-6-K3 muFc group, it does not appear to be a deleterious effect specific to the administration of PD1AB-6-K3 in the EAE model.

PD1AB-6-K3 muFc at 5 mg/kg significantly inhibited disease score on Day 20 to Day 31 (p<0.001-0.05; FIG. 31), and the AUC for disease score by 40% (p<0.05) compared to the dose-matched isotype control. PD1AB-6-K3 muFc or murine IgG2a at 1.25 mg/kg had no effect on disease score or AUC for disease score. The positive control FTY720 (fingolimod, Cayman Chemicals, Ann Arbor, Mich.) showed significant inhibition of EAE disease score from Day 20 until the last day of scoring of the study (Day 31; p<0.0001). In order to correlate serum levels of PD1AB-6-K3 muFc with effect on disease, serum was harvested at the termination of study and the levels of PD1AB-6-K3 muFc were measured. Very few animals had levels of PD1AB-6-K3 muFc above the level of detection (4 out of 10 animals at 5 mg/kg and only 1 animal out of 10 in the 1.25 mg/kg dose group). All the animals had measurable levels of anti-drug antibody. There was a trend toward reduction in human PD-1 expression levels in CD4+ and CD8+ T cells of spleen and blood after treatment with PD1AB-6-K3 muFc at 5 mg/kg compared to the dose matched isotype control group. At the termination of MOG-EAE experiments (Day 23 and Day 34), the spleen cells were tested in ex vivo assays. Splenocytes from the therapeutic PD1AB-6-K3 muFc group (Day 23 termination) showed significant (p<0.0001) inhibition of IFN-γ secretion (66%), a trend toward inhibition of IL-17 with high variability (40%), and significantly lower proliferation of CD4+ T cells in response to the highest dose of MOG in a recall assay (42%, FIG. 32A). Splenocytes from Day 34 did not show any effect on cytokine secretion or proliferation following PD1AB-6-K3 muFc treatment.

In summary, the in vitro and in vivo studies demonstrated that PD1AB-6-K3 is a high affinity PD-1 binding antibody that attenuates T cell activity as measured by the ability to inhibit the secretion of pro-inflammatory cytokines and to increase the expression of inhibitory receptors on T cells. The specificity and activity profile of PD1AB-6-K3 demonstrate the potential to be a beneficial therapeutic in autoimmune disease settings.

6. SEQUENCE LISTING

The present specification is being filed with a computer readable form (CRF) copy of the Sequence Listing. The CRF entitled 10624-385-999_SEQLIST.txt, which was created on Sep. 11, 2017 and is 50,882 bytes in size, is identical to the paper copy of the Sequence Listing and is incorporated herein by reference in its entirety. 

1.-108. (canceled)
 109. A method of managing, preventing, or treating an immune disorder in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that (A) (a) binds to an epitope of human PD-1 recognized by an antibody comprising a light chain variable region having an amino acid sequence of SEQ ID NO:8 and a heavy chain variable region having an amino acid sequence of SEQ ID NO:13; or (b) competes for the binding to human PD-1 with an antibody comprising a light chain variable region having an amino acid sequence of SEQ ID NO:8 and a heavy chain variable region having an amino acid sequence of SEQ ID NO:13; or (B) binds to PD-1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDR1), VL CDR2, and VL CDR3 of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 as set forth in Table 1; and/or (b) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1), VH CDR2, and VH CDR3 of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 as set forth in Table
 2. 110. The method of claim 109, wherein (i) the antibody or antigen-binding fragment thereof comprises: (a) a light chain variable region (VL) further comprising VL framework 1 (FR1), VL FR2, VL FR3, and VL FR4 of any one of antibodies PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 as set forth in Table 3; and/or (b) a heavy chain variable region (VH) further comprising VH framework 1 (FR1), VH FR2, VH FR3, and VH FR4 of any one of antibodies PD1 AB-1, PD1AB-2, PD1AB-3, PD1AB-4, PD1AB-5, or PD1AB-6 as set forth in Table 4; (ii) the VL CDR1, VL CDR2, and VL CDR3 that comprise amino acid sequences of SEQ ID NOS:1, 2, and 3, respectively, and the VH CDR1, VH CDR2, and VH CDR3 comprise amino acid sequences of SEQ ID NOS:4, 5, and 6, respectively; (iii) the VL CDR1, VL CDR2, and VL CDR3 that comprise amino acid sequences of SEQ ID NOS:7, 2, and 3, respectively, and the VH CDR1, VH CDR2, and VH CDR3 comprise amino acid sequences of SEQ ID NOS:4, 5, and 6, respectively; (iv) the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10; (v) the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; (vi) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:8; and (b) a VH comprising an amino acid sequence of SEQ ID NO:11; (vii) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:9; and (b) a VH comprising an amino acid sequence of SEQ ID NO:11; (viii) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:10; and (b) a VH comprising an amino acid sequence of SEQ ID NO:11; (ix) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:8; and (b) a VH comprising an amino acid sequence of SEQ ID NO:12; (x) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:9; and (b) a VH comprising an amino acid sequence of SEQ ID NO:12; (xi) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:10; and (b) a VH comprising an amino acid sequence of SEQ ID NO:12; (xii) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:8; and (b) a VH comprising an amino acid sequence of SEQ ID NO:13; (xiii) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:9; and (b) a VH comprising an amino acid sequence of SEQ ID NO:13; or (xiv) the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:10; and (b) a VH comprising an amino acid sequence of SEQ ID NO:13.
 111. The method of claim 109, wherein (i) the antibody or antigen-binding fragment thereof comprises a human IgG1 Fc region or a variant thereof; (ii) the antibody or antigen-binding fragment thereof comprises a human IgG1-K322A Fc region; (iii) the antibody or antigen-binding fragment thereof comprises a heavy chain Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:36-40; wherein optionally the antibody or antigen-binding fragment thereof further comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:41; or (iv) the antibody or antigen-binding fragment thereof comprises: (a) a light chain constant region comprising an amino acid sequence of SEQ ID NO:41; and (b) a heavy chain Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:36-40.
 112. The method of claim 109, wherein (i) the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO:31; (ii) the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:32; (iii) the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:32; (iv) wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:33; (v) the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:33; (vi) the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:34; (vii) the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:34; (viii) the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:35; or (ix) the antibody or antigen-binding fragment thereof comprises: (a) a light chain comprising an amino acid sequence of SEQ ID NO:31; and (b) a heavy chain comprising an amino acid sequence of SEQ ID NO:35.
 113. The method of claim 109, wherein, when bound to PD-1, the antibody or antigen-binding fragment binds to (i) at least one of residues 100-109 within an amino acid sequence of SEQ ID NO:42; wherein optionally, when bound to PD-1, the antibody or antigen-binding fragment binds to at least one of residues 100-105 within an amino acid sequence of SEQ ID NO:42; or (ii) at least one residue selected from the group consisting of N33, T51, S57, L100, N102, G103, R104, D105, H107, and S109 within an amino acid sequence of SEQ ID NO:42; wherein optionally when bound to PD-1, the antibody or antigen-binding fragment binds to (a) N33 within an amino acid sequence of SEQ ID NO:42; (b) T51 within an amino acid sequence of SEQ ID NO:42; (c) S57 within an amino acid sequence of SEQ ID NO:42; (d) L100 within an amino acid sequence of SEQ ID NO:42; (e) N102 within an amino acid sequence of SEQ ID NO:42; (f) G103 within an amino acid sequence of SEQ ID NO:42; (g) R104 within an amino acid sequence of SEQ ID NO:42; (h) D105 within an amino acid sequence of SEQ ID NO:42; (i) H107 within an amino acid sequence of SEQ ID NO:42; (j) S109 within an amino acid sequence of SEQ ID NO:42; or (k) G103 and R104 within an amino acid sequence of SEQ ID NO:42.
 114. The method of claim 109, wherein the antibody or antigen-binding fragment thereof specifically binds to human PD-1 and/or monkey PD-1, but not rodent PD-1; wherein optionally the K_(D) for binding to purified human PD-1 is from about 100 pM to about 10 nM, and the K_(D) for binding to human PD-1 expressed on cell surface and monkey PD-1 expressed on cell surface is from about 100 pM to about 10 nM.
 115. The method of claim 109, wherein the antibody or antigen-binding fragment thereof has attenuated ADCC activity and/or attenuated CDC activity.
 116. The method of claim 109, wherein the antibody or antigen-binding fragment thereof: (a) attenuates T cell activity; and/or (b) downregulates PD-1 expression on the surface of T cells.
 117. The method of claim 116, wherein the attenuation of T cell activity is measured by (i) inhibition of cytokine production; wherein optionally the cytokine that is inhibited by the antibody or antigen-binding fragment thereof comprises IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof; (ii) inhibition of T cell proliferation; (iii) upregulation of expression of an inhibitory receptor on the surface of T cells, wherein the inhibitory receptor is not PD-1; wherein optionally the inhibitory receptor is selected from the group consisting of LAG-3, CTLA-4, and TIM-3; (iv) inhibition of T cell recall response; (v) increase of T cell exhaustion; or (vi) increase of regulatory T cells.
 118. The method of claim 116, wherein the downregulation of PD-1 expression on the surface of T cells: (a) occurs as early as 4 hours after the treatment with the antibody or antigen-binding fragment thereof; and/or (b) is concurrent with or precedes cytokine inhibition.
 119. The method of claim 116, wherein (i) the EC₅₀ for attenuating T cell activity is from about 1 pM to about 10 pM, from about 10 pM to about 100 pM, from about 100 pM to about 1 nM, from about 1 nM to about 10 nM, or from about 10 nM to about 100 nM; (ii) the maximal percent attenuation of T cell activity is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%; or (iii) the maximal percent downregulation of PD-1 expression is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
 120. The method of claim 109, wherein (i) the antibody is a monoclonal antibody; (ii) the antibody is a humanized, human, or chimeric antibody; wherein optionally the humanized antibody is a deimmunized antibody or a composite human antibody; or (iii) the antibody or antigen-binding fragment thereof is a Fab, a Fab′, a F(ab′)₂, a Fv, a scFv, a dsFv, a diabody, a triabody, a tetrabody, or a multispecific antibody formed from antibody fragments.
 121. The method of claim 109, wherein the antibody or antigen-binding fragment thereof is conjugated to an agent; wherein optionally the agent is selected from the group consisting of a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.
 122. The method of claim 109, wherein the antibody or antigen-binding fragment thereof further comprises a pharmaceutically acceptable carrier.
 123. The method of claim 109, wherein the immune disorder is an inflammatory disease; wherein optionally the inflammatory disease is uveitis.
 124. The method of claim 109, wherein the immune disorder is an hypersensitivity disease; wherein optionally the hypersensitivity disease is selected from the group consisting of allergy, atopic dermatitis, and hypersensitivity vasculitis.
 125. The method of claim 109, wherein the immune disorder is an autoimmune disease; wherein optionally the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, multiple sclerosis, lupus, ankylosing spondylitis, type I diabetes, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, celiac disease, scleroderma, and temporal arteritis.
 126. The method of claim 125, wherein the lupus is systemic lupus erythematosus, cutaneous lupus erythematosus or lupus nephritis.
 127. The method of claim 109, wherein the immune cells in the subject express PD-1. 